Homogeneous preparations of il-28 and il-29

ABSTRACT

Homogeneous preparations of IL-28A, IL-28B, and IL-29 have been produced by mutating one or more of the cysteine residues in the polynucleotide sequences encoding the mature proteins. The cysteine mutant proteins can be shown to either bind to their cognate receptor or exhibit biological activity. One type of biological activity that is shown is an antiviral activity.

The present application is a divisional of U.S. patent application Ser.No. 10/914,772, filed Aug. 9, 2004, which claims the benefit of U.S.Patent Application Ser. Nos. 60/493,194, filed Aug. 7, 2003, 60/551,841,filed Mar. 10, 2004, and 60/559,142, filed Apr. 2, 2004, all of whichare herein incorporated by reference.

BACKGROUND OF THE INVENTION

Cytokines play important roles in the regulation of hematopoiesis andimmune responses, and can influence lymphocyte development. The humanclass II cytokine family includes interferon-α (IFN-α) subtypes,interferon-β (IFN-β), interferon-γ (IFN-γ), IL-10, IL-19 (U.S. Pat. No.5,985,614), MDA-7 (Jiang et al., Oncogene 11, 2477-2486, (1995)), IL-20(Jiang et al., Oncogene 11, 2477-2486, (1995)), IL-22 (Xie et al., J.Biol. Chem. 275, 31335-31339, (2000)), and AK-155 (Knappe et al., J.Virol. 74, 3881-3887, (2000)). Most cytokines bind and transduce signalsthrough either Class I or Class II cytokine receptors. Members of humanclass II cytokine receptor family include interferon-αR1 (IFN-αR1),interferon-γ-R2 (IFN-γ-R2), interferon-γ R1 (IFN-γ R1), interferon-γR2(IFN-γR2), IL-10R (Liu et al., J. Immunol. 152, 1821-1829, (1994)),CRF2-4 (Lutfalla et al. Genomics 16, 366-373, (1993)), IL-20Rβ (Blumberget al., Cell 104, 9-19, (2001)) (also known as zcytor7 (U.S. Pat. No.5,945,511) and CRF2-8 (Kotenko et al., Oncogene 19, 2557-2565, (2000)),IL-20Rβ (Blumberg et al., ibid, (2001)) (also known as DIRS1 (PCT WO99/46379)), IL-22RA1 (IL-22 receptor-α1, submitted to HUGO for approval)(also known as IL-22R (Xie et al., J. Biol. Chem. 275, 31335-31339,(2000)), zcytor11 (U.S. Pat. No. 5,965,704) and CRF2-9 (Kotenko et al.,Oncogene 19, 2557-2565, (2000)), and tissue factor.

Class II cytokine receptors are typically heterodimers composed of twodistinct receptor chains, the α and β receptor subunits (Stahl et al.,Cell 74, 587-590, (1993)). In general, the α subunits are the primarycytokine binding proteins, and the β subunits are required for formationof high affinity binding sites, as well as for signal transduction. Anexception is the IL-20 receptor in which both subunits are required forIL-20 binding (Blumberg et al., ibid, (2001)).

The class II cytokine receptors are identified by a conservedcytokine-binding domain of about 200 amino acids (D200) in theextracellular portion of the receptor. This cytokine-binding domain iscomprised of two fibronectin type III (FnIII) domains of approximately100 amino acids each (Bazan J. F. Proc. Natl. Acad. Sci. USA 87,6934-6938, (1990); Thoreau et al., FEBS Lett. 282, 16-31, (1991)). EachFnIII domain contains conserved Cys, Pro, and Trp residues thatdetermine a characteristic folding pattern of seven β-strands similar tothe constant domain of immunoglobulins (Uze et al., J. InterferonCytokine Res. 15, 3-26, (1995)). The conserved structural elements ofthe class II cytokine receptor family make it possible to identify newmembers of this family on the basis of primary amino acid sequencehomology.

The interleukins are a family of cytokines that mediate immunologicalresponses, including inflammation. Central to an immune response is theT cell, which produce many cytokines and adaptive immunity to antigens.Cytokines produced by the T cell have been classified as type 1 and type2 (Kelso, A. Immun. Cell Biol. 76:300-317, 1998). Type 1 cytokinesinclude IL-2, interferon-gamma (IFN-γ), LT-α, and are involved ininflammatory responses, viral immunity, intracellular parasite immunityand allograft rejection. Type 2 cytokines include IL-4, IL-5, IL-6,IL-10 and IL-13, and are involved in humoral responses, helminthimmunity and allergic response. Shared cytokines between Type 1 and 2include IL-3, GM-CSF and TNF-α. There is some evidence to suggest thatType 1 and Type 2 producing T cell populations preferentially migrateinto different types of inflamed tissue.

Of particular interest, from a therapeutic standpoint, are theinterferons (reviews on interferons are provided by De Maeyer and DeMaeyer-Guignard, “Interferons,” in The Cytokine Handbook, 3^(rd)Edition, Thompson (ed.), pages 491-516 (Academic Press Ltd. 1998), andby Walsh, Biopharmaceuticals: Biochemistry and Biotechnology, pages158-188 (John Wiley & Sons 1998)). Interferons exhibit a variety ofbiological activities, and are useful for the treatment of certainautoimmune diseases, particular cancers, and the enhancement of theimmune response against infectious agents, including viruses, bacteria,fungi, and protozoa. To date, six forms of interferon have beenidentified, which have been classified into two major groups. Theso-called “type I” IFNs include IFN-α, IFN-β, IFN-ω, IFN-δ, andinterferon-τ. Currently, IFN-γ and one subclass of IFN-α are the onlytype II IFNs.

Type I IFNs, which are thought to be derived from the same ancestralgene, have retained sufficient similar structure to act by the same cellsurface receptor. The α-chain of the human IFN-α/β receptor comprises anextracellular N-terminal domain, which has the characteristics of aclass II cytokine receptor. IFN-γ does not share significant homologywith the type I IFN or with the type II IFN-α subtype, but shares anumber of biological activities with the type I IFN.

Clinicians are taking advantage of the multiple activities ofinterferons by using the proteins to treat a wide range of conditions.For example, one form of IFN-α has been approved for use in more than 50countries for the treatment of medical conditions such as hairy cellleukemia, renal cell carcinoma, basal cell carcinoma, malignantmelanoma, AIDS-related Kaposi's sarcoma, multiple myeloma, chronicmyelogenous leukemia, non-Hodgkin's lymphoma, laryngeal papillomatosis,mycosis fungoides, condyloma acuminata, chronic hepatitis B, hepatitisC, chronic hepatitis D, and chronic non-A, non-B/C hepatitis. The U.S.Food and Drug Administration has approved the use of IFN-β to treatmultiple sclerosis, a chronic disease of the nervous system. IFN-γ isused to treat chronic granulomatous diseases, in which the interferonenhances the patient's immune response to destroy infectious bacterial,fungal, and protozoal pathogens. Clinical studies also indicate thatIFN-γ may be useful in the treatment of AIDS, leishmaniasis, andlepromatous leprosy.

IL-28A, IL-28B, and IL-29 comprise a recently discovered new family ofproteins that have sequence homology to type I interferons and genomichomology to IL-10. This new family is fully described in co-owned PCTapplication WO 02/086087 and Sheppard et al., Nature Immunol. 4:63-68,2003; both incorporated by reference herein. Functionally, IL-28 andIL-29 resemble type I INFs in their ability to induce an antiviral statein cells but, unlike type I IFNs, they do not display antiproliferativeactivity against certain B cell lines.

IL-28 and IL-29 are known to have an odd number of cysteines (PCTapplication WO 02/086087 and Sheppard et al., supra.) Expression ofrecombinant IL-28 and IL-29 can result in a heterogeneous mixture ofproteins composed of intramolecular disulfide bonding in multipleconformations. The separation of these forms can be difficult andlaborious. It is therefore desirable to provide IL-28 and IL-29molecules having a single intramolecular disulfide bonding pattern uponexpression and methods for refolding and purifying these preparations tomaintain homogeneity. Thus, the present invention provides forcompositions and methods to produce homogeneous preparations of IL-28and IL-29.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “level” when referring to immune cells, such as NK cells, Tcells, in particular cytotoxic T cells, B cells and the like, anincreased level is either increased number of cells or enhanced activityof cell function.

The term “level” when referring to viral infections refers to a changein the level of viral infection and includes, but is not limited to, achange in the level of CTLs or NK cells (as described above), a decreasein viral load, an increase antiviral antibody titer, decrease inserological levels of alanine aminotransferase, or improvement asdetermined by histological examination of a target tissue or organ.Determination of whether these changes in level are significantdifferences or changes is well within the skill of one in the art.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

“zcyto20”, “zcyto21”, “zcyto22” are the previous designations for humanIL-28A, human IL-29, and human IL-28B, respectively. The nucleotide andamino acid sequence for IL-28A are shown in SEQ ID NO:1 and SEQ ID NO:2,respectively. The nucleotide and amino acid sequences for IL-29 areshown in SEQ ID NO:3 and SEQ ID NO:4, respectively. The nucleotide andamino acid sequence for IL-28B are shown in SEQ ID NO:5 and SEQ ID NO:6,respectively. These sequences are fully described in PCT application WO02/086087 commonly assigned to ZymoGenetics, Inc., incorporated hereinby reference.

“zcyto24” and “zcyto25” are the previous designations for mouse IL-28,and are shown in SEQ ID NOs: 7, 8, 9, 10, respectively. Thepolynucleotide and polypeptides are fully described in PCT applicationWO 02/086087 commonly assigned to ZymoGenetics, Inc., incorporatedherein by reference.

“zcytor19” is the previous designation for IL-28 receptor α-subunit, andis shown in SEQ ID NO:11. The polynucleotides and polypeptides aredescribed in PCT application WO 02/20569 on behalf of Schering, Inc.,and WO 02/44209 assigned to ZymoGenetics, Inc and incorporated herein byreference. “IL-28 receptor” denotes the IL-28 α-subunit and CRF2-4subunit forming a heterodimeric receptor.

The present invention provides polynucleotide molecules, including DNAand RNA molecules, that encode Cysteine mutants of IL-28 and IL-29 thatresult in expression of a recombinant IL-28 or IL-29 preparation that isa homogeneous preparation. For the purposes of this invention, ahomogeneous preparation of IL-28 and IL-29 is a preparation in whichcomprises at least 98% of a single intramolecular disulfide bondingpattern in the purified polypeptide. In other embodiments, the singledisulfide conformation in a preparation of purified polypeptide is at99% homogeneous. In general, these Cysteine mutants will maintain somebiological activity of the wildtype IL-28 or IL-29, as described herein.For example, the molecules of the present invention can bind to theIL-28 receptor with some specificity. Generally, a ligand binding to itscognate receptor is specific when the K_(D) falls within the range of100 nM to 100 pM. Specific binding in the range of 100 mM to 10 nM K_(D)is low affinity binding. Specific binding in the range of 2.5 pM to 100pM K_(D) is high affinity binding. In another example, biologicalactivity of IL-28 or IL-29 Cysteine mutants is present when themolecules are capable of some level of antiviral activity associatedwith wildtype IL-28 or IL-29. Determination of the level of antiviralactivity is described in detail herein.

When referring to IL-28, the term shall mean both IL-28A and IL-28B.Previously IL-28A was designated zcyto20 (SEQ ID NOs:1 and 2), IL-29 wasdesignated zcyto21 (SEQ ID NOs:3 and 4), and IL-28B was designatedzcyto22 (SEQ ID NOs:5 and 6). (See, PCT application WO 02/086087 andSheppard et al., supra.) The mouse orthologs for IL-28 were previouslydesignated as zcyto24 (SEQ ID NOs:7 and 8), zcyto25 (SEQ ID NOs:9 and10).

Wildtype IL-28A gene encodes a polypeptide of 200 amino acids, as shownin SEQ ID NO:2. The signal sequence for IL-28A can be predicted ascomprising amino acid residue −25 (Met) through amino acid residue −1(Ala) of SEQ ID NO:2. The mature peptide for IL-28A begins at amino acidresidue 1 (Val) of SEQ ID NO:2. IL-28A helices are predicted as follow:helix A is defined by amino acid residues 31 (Ala) to 45 (Leu); helix Bby amino acid residues 58 (Thr) to 65 (Gln); helix C by amino acidresidues 69 (Arg) to 86 (Ala); helix D by amino acid residues 95 (Val)to 114 (Ala); helix E by amino acid residues 126 (Thr) to 142 (Lys); andhelix F by amino acid residues 148 (Cys) to 169 (Ala); as shown in SEQID NO: 2.

Wildtype IL-29 gene encodes a polypeptide of 200 amino acids, as shownin SEQ ID NO:4. The signal sequence for IL-29 can be predicted ascomprising amino acid residue −19 (Met) through amino acid residue −1(Ala) of SEQ ID NO:4, SEQ ID NO:1 19, or SEQ ID NO:121. The maturepeptide for IL-29 begins at amino acid residue 1 (Gly) of SEQ ID NO:4.IL-29 has been described in PCT application WO 02/02627. IL-29 helicesare predicted as follows: helix A is defined by amino acid residues 30(Ser) to 44 (Leu); helix B by amino acid residues 57 (Asn) to 65 (Val);helix C by amino acid residues 70 (Val) to 85 (Ala); helix D by aminoacid residues 92 (Glu) to 111 (Gln); helix E by amino acid residues 118(Thr) to 139 (Lys); and helix F by amino acid residues 144 (Gly) to 170(Leu); as shown in SEQ ID NO:4.

Wildtype IL-28B gene encodes a polypeptide of 200 amino acids, as shownin SEQ ID NO:6. The signal sequence for IL-28B can be predicted ascomprising amino acid residue −21 (Met) through amino acid residue −1(Ala) of SEQ ID NO:6. The mature peptide for IL-28B begins at amino acidresidue 1 (Val) of SEQ ID NO:6. IL-28B helices are predicted as follow:helix A is defined by amino acid residues 31 (Ala) to 45 (Leu); helix Bby amino acid residues 58 (Thr) to 65 (Gln); helix C by amino acidresidues 69 (Arg) to 86 (Ala); helix D by amino acid residues 95 (Gly)to 114 (Ala); helix E by amino acid residues 126 (Thr) to 142 (Lys); andhelix F by amino acid residues 148 (Cys) to 169 (Ala); as shown in SEQID NO:6.

The present invention provides mutations in the IL-28 and IL-29 wildtypesequences as shown in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, that result inexpression of single forms of the IL-28 or IL-29 molecule. Because theheterogeneity of forms is believed to be a result of multipleintramolecular disulfide bonding patterns, specific embodiments of thepresent invention includes mutations to the cysteine residues within thewildtype IL-28 and IL-29 sequences. When IL-28 and IL-29 are expressedin E. coli, an N-terminal or amino-terminal Methionine is present. SEQID NOs:12-17, for example, show the nucleotide and amino acid residuenumbering for IL-28A, IL-29 and IL-28B when the N-terminal Met ispresent. Table 1 shows the possible combinations of intramoleculardisulfide bonded cysteine pairs for wildtype IL-28A, IL-28B, and IL-29.TABLE 1 IL-28A C₁₆- C₄₈- C₅₀- C₁₆₇- C₁₆- C₁₆- C₄₈- C₅₀- C₁₁₅- SEQ IDNO:2 C₁₁₅ C₁₄₈ C₁₄₈ C₁₇₄ C₄₈ C₅₀ C₁₁₅ C₁₁₅ C₁₄₈ Met IL-28A C₁₇- C₄₉-C₅₁- C₁₆₈- C₁₇- C₁₇- C₄₉- C₅₁- C₁₁₆- SEQ ID NO:13 C₁₁₆ C₁₄₉ C₁₄₉₈ C₁₇₅C₄₉ C₅₁ C₁₁₆ C₁₁₆ C₁₄₉ IL-29 C₁₅- C₄₉- C₁₁₂- SEQ ID NO:4 C₁₁₂ C₁₄₅ C₁₇₁Met IL-29 C₁₆- C₅₀- C₁₁₃- SEQ ID NO:15 C₁₁₃ C₁₄₆ C₁₇₂ IL-28B C₁₆- C₄₈-C₅₀- C₁₆₇- C₁₆- C₁₆- C₄₈- C₅₀- C₁₁₅- SEQ ID NO:6 C₁₁₅ C₁₄₈ C₁₄₈ C₁₇₄ C₄₈C₅₀ C₁₁₅ C₁₁₅ C₁₄₈ Met IL-28B C₁₇- C₄₉- C₅₁- C₁₆₈- C₁₇- C₁₇- C₄₉- C₅₁-C₁₁₆- SEQ ID NO:17 C₁₁₆ C₁₄₉ C₁₄₉ C₁₇₅ C₄₉ C₅₁ C₁₁₆ C₁₁₆ C₁₄₉

The polynucleotide and polypeptide molecules of the present inventionhave a mutation at one or more of the Cysteines present in the wildtypeIL-28A, IL-29 or IL-28B molecules, yet retain some biological activityas described herein. Table 2 illustrates exemplary Cysteine mutants, inparticular point mutations of cysteine (C) to serine (S). TABLE 2 IL-28AC48S SEQ ID NO:19 Met IL-28A C49S SEQ ID NO:21 IL-28A C50S SEQ ID NO:23Met IL-28A C51S SEQ ID NO:25 IL-29 C171S SEQ ID NO:27 Met IL-29 C172SSEQ ID NO:29

All the members of the family have been shown to bind to the same classTI cytokine receptor, IL-28R. IL-28 α-subunit was previously designatedzcytor19 receptor. While not wanting to be bound by theory, thesemolecules appear to all signal through IL-28R receptor via the samepathway. IL-28 receptor is described in a commonly assigned PCT patentapplication WO 02/44209, incorporated by reference herein; Sheppard etal., supra; Kotenko et al., Nature Immunol. 4:69-77, 2003; and PCTWO/03/040345. IL-28R is a member of the Class II cytokine receptorswhich is characterized by the presence of one or more cytokine receptormodules (CRM) in their extracellular domains. Other class II cytokinereceptors include zcytor11 (commonly owned U.S. Pat. No. 5,965,704),CRF2-4 (Genbank Accession No. Z17227), IL-10R (Genbank Accession No.sU00672 and NM_(—)001558), DIRS1, zcytor7 (commonly owned U.S. Pat. No.5,945,511), and tissue factor. IL-28 receptor, like all known class IIreceptors except interferon-alpha/beta receptor alpha chain, has only asingle class II CRM in its extracellular domain.

Four-helical bundle cytokines are also grouped by the length of theircomponent helices. “Long-helix” form cytokines generally consist ofbetween 24-30 residue helices, and include IL-6, ciliary neutrotrophicfactor (CNTF), leukemia inhibitory factor (LIF) and human growth hormone(hGH). “Short-helix” form cytokines generally consist of between 18-21residue helices and include IL-2, IL-4 and GM-CSF. Studies using CNTFand IL-6 demonstrated that a CNTF helix can be exchanged for theequivalent helix in IL-6, conferring CTNF-binding properties to thechimera. Thus, it appears that functional domains of four-helicalcytokines are determined on the basis of structural homology,irrespective of sequence identity, and can maintain functional integrityin a chimera (Kallen et al., J. Biol. Chem. 274:11859-11867, 1999).Therefore, Cysteine mutants IL-28 and IL-29 polypeptides will be usefulfor preparing chimeric fusion molecules, particularly with otherinterferons to determine and modulate receptor binding specificity. Ofparticular interest are fusion proteins that combine helical and loopdomains from interferons and cytokines such as INF-60 , IL-10, humangrowth hormone.

The present invention provides polynucleotide molecules, including DNAand RNA molecules, that encode, for example, Cysteine mutant IL-28 orIL-29 polypeptides. For example, the present invention providesdegenerate nucleotide sequences encoding IL-28A C48S, Met IL-28A C49S,IL-28A C50S, Met IL-28A C51S, IL-29 C171S and Met IL-29 C172Spolypeptides disclosed herein. Those skilled in the art will readilyrecognize that, in view of the degeneracy of the genetic code,considerable sequence variation is possible among these polynucleotidemolecules. SEQ ID NOs:30, 31, 32, 33, 34, and 35 are a degenerate DNAsequences that encompasses all DNAs that encode IL-28A C48S, Met IL-28AC49S, IL-28A C50S, Met IL-28A C51S, IL-29 C171S and Met Met IL-29 C172S,respectively. Those skilled in the art will recognize that thedegenerate sequence of SEQ ID NOs: 30, 31, 32, 33, 34, and 35 alsoprovides all RNA sequences encoding SEQ ID NOs: 30, 31, 32, 33, 34, and35 by substituting U for T and are thus comtemplated by the presentinvention.

IL-28A polypeptides of the present invention also include a mutation atthe second cysteine, C2, of the mature polypeptide. For example, C2 fromthe N-terminus or amino-terminus of the polypeptide of SEQ ID NO:2 isthe cysteine at amino acid position 48, or position 49 (additionalN-terminal Met) if expressed in E coli (see, for example, SEQ ID NO:13).This second cysteine (of which there are seven, like IL-28B) or C2 ofIL-28A can be mutated, for example, to a serine, alanine, threonine,valine, or asparagine. IL-28A C2 mutant molecules of the presentinvention include, for example, polynucleotide molecules as shown in SEQID NOs:20 and 22, including DNA and RNA molecules, that encode IL-28A C2mutant polypeptides as shown in SEQ ID NOs:21 and 23, respectively. SEQID NOs:36 and 37 are additional IL-28A C2 polypeptides of the presentinvention.

In addition to the IL-28A C2 mutants, the present invention alsoincludes IL-28A polypeptides comprising a mutation at the third cysteineposition, C3, of the mature polypeptide. For example, C3 from theN-terminus or amino-terminus of the polypeptide of SEQ ID NO:2, is thecysteine at position 50, or position 51 (additional N-terminal Met) ifexpressed in E. coli (see, for example, SEQ ID NO:13). IL-28A C3 mutantmolecules of the present invention include, for example, polynucleotidemolecules as shown in SEQ ID NOs:24 and 26, including DNA and RNAmolecules, that encode IL-28A C3 mutant polypeptides as shown in SEQ IDNOs:25 and 27, respectively. SEQ ID NOs:38 and 39 are additional IL-28AC3 polypeptides of the present invention.

The IL-28A polypeptides of the present invention include, for example,SEQ ID NOs:2, 13, 19, 21, 23, and 25, which are encoded by IL-28Apolynucleotide molecules as shown in SEQ ID NOs:1, 12, 18, 20, 22, and24, respectively. Further IL-28A polypeptides of the present inventioninclude, for example, SEQ ID NOs:36, 37, 38, and 39.

IL-28B polypeptides of the present invention also include a mutation atthe second cysteine, C2, of the mature polypeptide. For example, C2 fromthe N-terminus or amino-terminus of the polypeptide of SEQ ID NO:6 isthe cysteine at amino acid position 48, or position 49 (additionalN-terminal Met) if expressed in E coli (see, for example, SEQ ID NO:17).This second cysteine (of which there are seven, like IL-28A) or C2 ofIL-28B can be mutated, for example, to a serine, alanine, threonine,valine, or asparagine. IL-28B C2 mutant molecules of the presentinvention include, for example, polynucleotide molecules as shown in SEQID NOs:122 and 124, including DNA and RNA molecules, that encode IL-28BC2 mutant polypeptides as shown in SEQ ID NOs:123 and 125, respectively.Additional IL-28B C2 mutant molecules of the present invention includepolynucleotide molecules as shown in SEQ ID NOs:130 and 132 includingDNA and RNA molecules, that encode IL-28B C2 mutant polypeptides asshown in SEQ ID NOs:131 and 133, respectively (PCT publication WO03/066002 (Kotenko et al.)).

In addition to the IL-28B C2 mutants, the present invention alsoincludes IL-28B polypeptides comprising a mutation at the third cysteineposition, C3, of the mature polypeptide. For example, C3 from theN-terminus or amino-terminus of the polypeptide of SEQ ID NO:6, is thecysteine at position 50, or position 51 (additional N-terminal Met) ifexpressed in E. coli (see, for example, SEQ ID NO:17). IL-28B C3 mutantmolecules of the present invention include, for example, polynucleotidemolecules as shown in SEQ ID NOs:126 and 128, including DNA and RNAmolecules, that encode IL-28B C3 mutant polypeptides as shown in SEQ IDNOs:127 and 129, respectively. Additional IL-28B C3 mutant molecules ofthe present invention include polynucleotide molecules as shown in SEQID NOs:134 and 136 including DNA and RNA molecules, that encode IL-28BC3 mutant polypeptides as shown in SEQ ID NOs:135 and 137, respectively(PCT publication WO 03/066002 (Kotenko et al.)).

The IL-28B polypeptides of the present invention include, for example,SEQ ID NOs:6, 17, 123, 125, 127, 129, 131, 133, 135, and 137, which areencoded by IL-28B polynucleotide molecules as shown in SEQ ID NOs:5, 16,122, 124, 126, 128, 130, 132, 134, and 136, respectively.

IL-29 polypeptides of the present invention also include, for example, amutation at the fifth cysteine, C5, of the mature polypeptide. Forexample, C5 from the N-terminus of the polypeptide of SEQ ID NO:4, isthe cysteine at position 171, or position 172 (additional N-terminalMet) if expressed in E. coli. (see, for example, SEQ ID NO:15). Thisfifth cysteine or C5 of IL-29 can be mutated, for example, to a serine,alanine, threonine, valine, or asparagine. These IL-29 C5 mutantpolypeptides have a disulfide bond pattern of C1(Cys15 of SEQ IDNO:4)/C3(Cys112 of SEQ ID NO:4) and C2(Cys49 of SEQ ID NO:4)/C4(Cys145of SEQ ID NO:4). Additional IL-29 C5 mutant molecules of the presentinvention include polynucleotide molecules as shown in SEQ ID NOs:26,28, 82, 84, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, and160, including DNA and RNA molecules, that encode IL-29 C5 mutantpolypeptides as shown in SEQ ID NOs:27, 29, 83, 85, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, and 161, respectively. AdditionalIL-29 C5 mutant molecules of the present invention includepolynucleotide molecules as shown in SEQ ID NOs:86, 88, 94, and 96,including DNA and RNA molecules, that encode IL-29 C5 mutantpolypeptides as shown in SEQ ID NOs:87, 89, 95, and 97, respectively(PCT publication WO 03/066002 (Kotenko et al.)). Additional, IL-29 C5mutant molecules of the present invention include polynucleotidemolecules as shown in SEQ ID NOs:102, 104, 110, and 112, including DNAand RNA molecules, that encode IL-29 C5 mutant polypeptides as shown inSEQ ID NOs:103, 105, 111, and 113, respectively (PCT publication WO02/092762 (Baum et al.)).

In addition to the IL-29 C5 mutants, the present invention also includesIL-29 polypeptides comprising a mutation at the first cysteine position,C1, of the mature polypeptide. For example, C1 from the N-terminus ofthe polypeptide of SEQ ID NO:4, is the cysteine at position 15, orposition 16 (additional N-terminal Met) if expressed in E. coli (see,for example, SEQ ID NO:15). These IL-29 C1 mutant polypeptides will thushave a predicted disulfide bond pattern of C2(Cys49 of SEQ IDNO:4)/C4(Cys145 of SEQ ID NO:4) and C3(Cys112 of SEQ ID NO:4)/C5(Cys171of SEQ ID NO:4). Additional IL-29 C1 mutant molecules of the presentinvention include polynucleotide molecules as shown in SEQ ID NOs:74,76, 78, and 80, including DNA and RNA molecules, that encode IL-29 C1mutant polypeptides as shown in SEQ ID NOs:75, 77, 79 and 81,respectively. Additional IL-29 C1 mutant molecules of the presentinvention include polynucleotide molecules as shown in SEQ ID NOs:90,92, 98, and 100, including DNA and RNA molecules, that encode IL-29 C1mutant polypeptides as shown in SEQ ID NOs:91, 93, 99, and 101,respectively (PCT publication WO 03/066002 (Kotenko et al.)).Additional, IL-29 C1 mutant molecules of the present invention includepolynucleotide molecules as shown in SEQ ID NOs:106, 108, 114, and 116,including DNA and RNA molecules, that encode IL-29 C1 mutantpolypeptides as shown in SEQ ID NOs:107, 109, 115, and 117, respectively(PCT publication WO 02/092762 (Baum et al.)).

The IL-29 polypeptides of the present invention, for example, SEQ IDNOs:4, 15, 27, 29, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, and 161, which are encoded by IL-29polynucleotide molecules as shown in SEQ ID NOs:3, 14, 26, 28, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, and 160, may further include a signal sequence as shown in SEQ IDNO:119 or a signal sequence as shown in SEQ ID NO:121. In addition, thepresent invention also includes the IL-29 polypeptides as shown in SEQID NOs:40 and 41. A polynucleotide molecule encoding the signal sequencepolypeptide of SEQ ID NO:119 is shown as SEQ ID NO:118. A polynucleotidemolecule encoding the signal sequence polypeptide of SEQ ID NO:120 isshown as SEQ ID NO:121.

Within one aspect the present invention provides an isolated polypeptidecomprising a sequence having at least 90 percent or 95 percent sequenceidentity to a sequence of amino acid residues selected from the groupconsisting of SEQ ID NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27,29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, and 161. The polypeptide may optionally comprise at least 15, atleast 30, at least 45, or at least 60 sequential amino acids of an aminoacid sequence as shown in SEQ ID NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21,23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123,125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,153, 155, 157, 159, and 161. In another embodiment, the isolatedpolypeptide is an amino acid residues are selected from the groupconsisting of SEQ ID NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27,29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, and 161. The polypeptide may have a conservative amino acid change,compared with the amino acid sequence selected from the group consistingof SEQ ID NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36,37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131,133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,and 161.

Within a another aspect the present invention provides a fusion proteincomprising a polypeptide that comprises a sequence of amino acidresidues selected from the group consisting of SEQ ID NOs:2, 4, 6, 8,10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, and 161; and a polyalkyloxide moiety. The polyalkyl oxcide moiety may optionally be polyethyleneglycol, such as a 20 kD mPEG propionaldehyde or a 30 kD mPEGpropionaldehyde. The polyethylene glycol may be linear or branched. Thepolyethylene glycol may be covalently attached N-terminally orC-terminally to the polypeptide.

Within a another aspect the present invention provides a fusion proteincomprising a first polypeptide and a second polypeptide joined by apeptide bond, wherein the first polypeptide comprises a sequence ofamino acid residues selected from the group consisting of SEQ ID NOs:2,4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161; and a secondpolypeptide. The second polypeptide may optionally be an antibodyfragment. The antibody fragment may optionally be F(ab′), F(ab), Fab′,Fab, Fv, scFv, and/or minimal recognition unit. The second polypeptidemay optionally be human albumin. The second polypeptide may optionallybe a polypeptide selected from the group consisting of affinity tags,toxins, radionucleotides, enzymes and fluorophores.

Table 3 sets forth the one-letter codes used within SEQ ID NOs:30, 31,32, 33, 34, and 35 to denote degenerate nucleotide positions.“Resolutions” are the nucleotides denoted by a code letter. “Complement”indicates the code for the complementary nucleotide(s). For example, thecode Y denotes either C or T, and its complement R denotes A or G, withA being complementary to T, and G being complementary to C. TABLE 3Nucleotide Resolution Complement Resolution A A T T C C G G G G C C T TA A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T WA|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T NA|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NOs:30, 31, 32, 33, 34, and 35,encompassing all possible codons for a given amino acid, are set forthin Table 4. TABLE 4 One Amino Letter Degenerate Acid Code Codons CodonCys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACGACT ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGAGGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GARGln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGNLys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTGCTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TACTAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SARAny X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence, for example, of SEQ ID NOs:19, 21, 23, 25, 27, 29, 36, 37, 38,39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and161. Variant sequences can be readily tested for functionality asdescribed herein.

One of ordinary skill in the art will also appreciate that differentspecies can exhibit “preferential codon usage.” In general, see,Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 4). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNOs:30, 31, 32, 33, 34, and 35 serves as a template for optimizingexpression of polynucleotides in various cell types and species commonlyused in the art and disclosed herein. Sequences containing preferentialcodons can be tested and optimized for expression in various species,and tested for functionality as disclosed herein.

Within another aspect, the present invention provides an isolatedpolynucleotide selected from the group consisting of SEQ ID NOs:1, 3, 5,7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, and 160.

Within another aspect, the present invention provides an isolatedpolynucleotide capable of hybridizing to a sequence selected from thegroup consisting of SEQ ID NOs:1, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22,24, 26, 28, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, and160, or a complement thereof, under hybridization conditions of 50%formamide, 5×SSC (1×SSC: 0.15 M sodium chloride and 15 mM sodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution (100×Denhardt's solution: 2% (w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone,and 2% (w/v) bovine serum albumin, 10% dextran sulfate, and 20 mg/mldenatured, sheared salmon sperm DNA at about 42° C. to about 70° C.,wherein the isolated polynucleotide encodes a polypeptide havingantiviral activity. Optionally, the encoded polypeptide has antiviralactivity to hepatitis B and/or hepatitis C. Optionally, the isolatedpolynucleotide may encode at least a portion of a sequence selected fromthe group of SEQ ID NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27,29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, and 161. The isolated polynucleotide may encode a polypeptiderepresented by SEQ ID NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25,27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, or 161.

In another aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide wherein the encoded polypeptide isselected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 13, 15,17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, and 161.

In another aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide wherein the encoded polypeptidehas at least 90 percent or 95 percent sequence identity to a sequenceselected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 13, 15,17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, and 161, wherein the encoded polypeptidehas antiviral activity. Optionally, the encoded polypeptide hasantiviral activity to hepatitis B and/or hepatitis C.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of Cysteine mutant IL-28 or IL-29 RNA. Suchtissues and cells are identified by Northern blotting (Thomas, Proc.Natl. Acad. Sci. USA 77:5201, 1980), or by screening conditioned mediumfrom various cell types for activity on target cells or tissue. Once theactivity or RNA producing cell or tissue is identified, total RNA can beprepared using guanidinium isothiocyanate extraction followed byisolation by centrifugation in a CsC1 gradient (Chirgwin et al.,Biochemistry 18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNAusing the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA69:1408-12, 1972). Complementary DNA (cDNA) is prepared from poly(A)⁺RNA using known methods. In the alternative, genomic DNA can beisolated. Polynucleotides encoding Cysteine mutant IL-28 or IL-29polypeptides are then identified and isolated by, for example,hybridization or PCR.

A full-length clones encoding Cysteine mutant IL-28 or IL-29 can beobtained by conventional cloning procedures. Complementary DNA (cDNA)clones are preferred, although for some applications (e.g., expressionin transgenic animals) it may be preferable to use a genomic clone, orto modify a cDNA clone to include at least one genomic intron. Methodsfor preparing cDNA and genomic clones are well known and within thelevel of ordinary skill in the art, and include the use of the sequencedisclosed herein, or parts thereof, for probing or priming a library.Expression libraries can be probed with antibodies to IL-28 receptorfragments, or other specific binding partners.

Those skilled in the art will recognize that the sequence disclosed in,for example, SEQ ID NOs:1, 3, and 5, respectively, represent mutationsof single alleles of human IL-28 and IL-29 bands, and that allelicvariation and alternative splicing are expected to occur. For example,an IL-29 variant has been identified where amino acid residue 169 (Asn)as shown in SEQ ID NO:4 is an Arg residue, as described in WO 02/086087.Such allelic variants are included in the present invention. Allelicvariants of this sequence can be cloned by probing cDNA or genomiclibraries from different individuals according to standard procedures.Allelic variants of the DNA sequence shown in SEQ ID NO:1, 3 and 5,including those containing silent mutations and those in which mutationsresult in amino acid sequence changes, in addition to the cysteinemutations, are within the scope of the present invention, as areproteins which are allelic variants of SEQ ID NOs:2, 4, and 6. cDNAsgenerated from alternatively spliced mRNAs, which retain the propertiesof Cysteine mutant IL-28 or IL-29 polypeptides, are included within thescope of the present invention, as are polypeptides encoded by suchcDNAs and mRNAs. Allelic variants and splice variants of these sequencescan be cloned by probing cDNA or genomic libraries from differentindividuals or tissues according to standard procedures known in theart, and mutations to the polynucleotides encoding cysteines or cysteineresidues can be introduced as described herein.

Within embodiments of the invention, isolated variant or Cysteine mutantIL-28- and IL-29-encoding nucleic acid molecules can hybridize understringent conditions to nucleic acid molecules having the nucleotidesequence of SEQ ID NOs:18, 20, 22, 24, 26, 28, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, and 160 or to nucleic acid molecules having anucleotide sequence complementary to SEQ ID NOs:18, 20, 22, 24, 26, 28,74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, and 160. In general,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases.

It is well within the abilities of one skilled in the art to adapt theseconditions for use with a particular polynucleotide hybrid. The T_(m)for a specific target sequence is the temperature (under definedconditions) at which 50% of the target sequence will hybridize to aperfectly matched probe sequence. Those conditions which influence theT_(m) include, the size and base pair content of the polynucleotideprobe, the ionic strength of the hybridization solution, and thepresence of destabilizing agents in the hybridization solution. Numerousequations for calculating T_(m) are known in the art, and are specificfor DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences ofvarying length (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor Press 1989);Ausubel et al., (eds.), Current Protocols in Molecular Biology (JohnWiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to MolecularCloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such asOLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0 (PremierBiosoft International; Palo Alto, Calif.), as well as sites on theInternet, are available tools for analyzing a given sequence andcalculating T_(m) based on user defined criteria. Such programs can alsoanalyze a given sequence under defined conditions and identify suitableprobe sequences. Typically, hybridization of longer polynucleotidesequences, >50 base pairs, is performed at temperatures of about 20-25°C. below the calculated T_(m). For smaller probes, <50 base pairs,hybridization is typically carried out at the T_(m) or 5-10° C. belowthe calculated T_(m). This allows for the maximum rate of hybridizationfor DNA-DNA and DNA-RNA hybrids.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×-2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55-65° C. That is, nucleic acid moleculesencoding a variant or Cysteine mutant IL-28 or IL-29 polypeptideshybridize with a nucleic acid molecule having the nucleotide sequence ofSEQ ID NOs:18, 20, 22, 24, 26, 28, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, and 160, respectively (or its complement) under stringentwashing conditions, in which the wash stringency is equivalent to0.5×-2×SSC with 0.1% SDS at 55-65° C., including 0.5×SSC with 0.1% SDSat 55° C., or 2×SSC with 0.1% SDS at 65° C. One of skill in the art canreadily devise equivalent conditions, for example, by substituting SSPEfor SSC in the wash solution.

Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. In other words, nucleic acid molecules encoding a variant of aCysteine mutant IL-28 or IL-29 polypeptide hybridize with a nucleic acidmolecule having the nucleotide sequence of SEQ ID NOs:18, 20, 22, 24,26, 28, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 122, 124, 126, 128, 130, 132, 134,136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, and 160 (orits complement) under highly stringent washing conditions, in which thewash stringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65°C., including 0.1×SSC with 0.1% SDS at 50° C., or 0.2×SSC with 0.1% SDSat 65° C.

The present invention also provides isolated IL-28 or IL-29 polypeptidesthat have a substantially similar sequence identity to the polypeptidesof the present invention, for example SEQ ID NOs:2, 4, 6, 8, 10, 13, 15,17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159 or 161. The term “substantially similarsequence identity” is used herein to denote polypeptides comprising atleast 80%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or greater than 99% sequence identity to the sequences shownin SEQ ID NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36,37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159 or 161, or their orthologs. The present invention also includespolypeptides that comprise an amino acid sequence having at least 80%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, orgreater than 99% sequence identity to a polypeptide or fragment thereofof the present invention. The present invention further includespolynucleotides that encode such polypeptides. The IL-28 and IL-29polypeptides of the present invention are preferably recombinantpolypeptides. In another aspect, the IL-28 and IL-29 polypeptides of thepresent invention have at least 15, at least 30, at least 45, or atleast 60 sequential amino acids. For example, an IL-28 or IL-29polypeptide of the present invention relates to a polypeptide having atleast 15, at least 30, at least 45, or at least 60 sequential aminoacids from SEQ ID NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27,29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159 or 161. Methods for determining percent identity are describedbelow.

The present invention also contemplates variant nucleic acid moleculesthat can be identified using two criteria: a determination of thesimilarity between the encoded polypeptide with the amino acid sequenceof SEQ ID NOs:19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159 or 161 respectively, and/ora hybridization assay, as described above. Such variants include nucleicacid molecules: (1) that hybridize with a nucleic acid molecule havingthe nucleotide sequence of SEQ ID NOs:18, 20, 22, 24, 26, 28, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, and 160, respectively (orits complement) under stringent washing conditions, in which the washstringency is equivalent to 0.5×-2×SSC with 0.1% SDS at 55-65° C.; or(2) that encode a polypeptide having at least 80%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or greater than 99%sequence identity to the amino acid sequence of SEQ ID NOs:19, 21, 23,25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159 or 161. Alternatively, variants can be characterized asnucleic acid molecules: (1) that hybridize with a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NOs:18, 20, 22, 24, 26, 28, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, or 160, respectively (orits complement) under highly stringent washing conditions, in which thewash stringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65°C.; and (2) that encode a polypeptide having at least 80%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or greater than 99%sequence identity to the amino acid sequence of SEQ ID NOs:19, 21, 23,25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159 or 161, respectively.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 4 (amino acids are indicated by the standard one-lettercodes).Total number of identical matches ______×100 [length of the longersequence plus the number of gaps introduced into the longer sequence inorder to align the two sequences]

TABLE 5 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant IL-28 or IL-29. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:2) and a testsequence that have either the highest density of identities (if the ktupvariable is 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then rescored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62. These parameters can be introducedinto a FASTA program by modifying the scoring matrix file (“SMATRIX”),as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdefault.

Variant IL-28 or IL-29 Cysteine mutant polypeptides or polypeptides withsubstantially similar sequence identity are characterized as having oneor more amino acid substitutions, deletions or additions. These changesare preferably of a minor nature, that is conservative amino acidsubstitutions (see Table 6) and other substitutions that do notsignificantly affect the folding or activity of the polypeptide; smalldeletions, typically of one to about 30 amino acids; and amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue, a small linker peptide of up to about 20-25 residues, or anaffinity tag. The present invention thus includes polypeptides of fromabout 146 to 207 amino acid residues that comprise a sequence having atleast 80%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least least 96%, at least 97%, at least 98%,at least 99%, or greater than 99% sequence identity to the correspondingregion of SEQ ID NOs:19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159 or 161. Polypeptidescomprising affinity tags can further comprise a proteolytic cleavagesite between the IL-28 and IL-29 polypeptide and the affinity tag.Preferred such sites include thrombin cleavage sites and factor Xacleavage sites. TABLE 6 Conservative amino acid substitutions Basic:arginine lysine histidine Acidic: glutamic acid aspartic acid Polar:glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

Determination of amino acid residues that comprise regions or domainsthat are critical to maintaining structural integrity can be determined.Within these regions one can determine specific residues that will bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to alignment of multiple sequences withhigh amino acid or nucleotide identity, secondary structurepropensities, binary patterns, complementary packing and buried polarinteractions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 andCordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general,when designing modifications to molecules or identifying specificfragments determination of structure will be accompanied by evaluatingactivity of modified molecules.

Amino acid sequence changes are made in Cysteine mutant IL-28 or IL-29polypeptides so as to minimize disruption of higher order structureessential to biological activity. For example, where the Cysteine mutantIL-28 or IL-29 polypeptide comprises one or more helices, changes inamino acid residues will be made so as not to disrupt the helix geometryand other components of the molecule where changes in conformation abatesome critical function, for example, binding of the molecule to itsbinding partners. The effects of amino acid sequence changes can bepredicted by, for example, computer modeling as disclosed above ordetermined by analysis of crystal structure (see, e.g., Lapthorn et al.,Nat. Struct. Biol. 2:266-268, 1995). Other techniques that are wellknown in the art compare folding of a variant protein to a standardmolecule (e.g., the native protein). For example, comparison of thecysteine pattern in a variant and standard molecules can be made. Massspectrometry and chemical modification using reduction and alkylationprovide methods for determining cysteine residues which are associatedwith disulfide bonds or are free of such associations (Bean et al.,Anal. Biochem. 201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993;and Patterson et al., Anal. Chem. 66:3727-3732, 1994). It is generallybelieved that if a modified molecule does not have the same cysteinepattern as the standard molecule folding would be affected. Another wellknown and accepted method for measuring folding is circular dichrosism(CD). Measuring and comparing the CD spectra generated by a modifiedmolecule and standard molecule is routine (Johnson, Proteins 7:205-214,1990). Crystallography is another well known method for analyzingfolding and structure. Nuclear magnetic resonance (NMR), digestivepeptide mapping and epitope mapping are also known methods for analyzingfolding and structurally similarities between proteins and polypeptides(Schaanan et al., Science 257:961-964, 1992).

A Hopp/Woods hydrophilicity profile of the Cysteine mutant IL-28 orIL-29 protein sequence as shown in SEQ ID NOs:19, 21, 23, 25, 27, 29,36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159 or 161 can be generated (Hopp et al., Proc. Natl. Acad.Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquieret al., Protein Engineering 11:153-169, 1998). The profile is based on asliding six-residue window. Buried G, S, and T residues and exposed H,Y, and W residues were ignored. Those skilled in the art will recognizethat hydrophilicity or hydrophobicity will be taken into account whendesigning modifications in the amino acid sequence of a Cysteine mutantIL-28 or IL-29 polypeptide, so as not to disrupt the overall structuraland biological profile. Of particular interest for replacement arehydrophobic residues selected from the group consisting of Val, Leu andIle or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp.

The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between IFN-α and members of the familyof IL-28A, IL-28B, and IL-29 (as shown in Tables 1 and 2). Using methodssuch as “FASTA” analysis described previously, regions of highsimilarity are identified within a family of proteins and used toanalyze amino acid sequence for conserved regions. An alternativeapproach to identifying a variant polynucleotide on the basis ofstructure is to determine whether a nucleic acid molecule encoding apotential variant IL-28 or IL-29 gene can hybridize to a nucleic acidmolecule as discussed above.

Other methods of identifying essential amino acids in the polypeptidesof the present invention are procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Natl Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant Cysteine mutant molecules are tested forbiological or biochemical activity as disclosed below to identify aminoacid residues that are critical to the activity of the molecule. Seealso, Hilton et al., J. Biol. Chem. 271:4699 (1996).

The present invention also includes functional fragments of Cysteinemutant IL-28 or IL-29 polypeptides and nucleic acid molecules encodingsuch functional fragments. A “functional” Cysteine mutant IL-28 or IL-29or fragment thereof as defined herein is characterized by itsproliferative or differentiating activity, by its ability to induce orinhibit specialized cell functions, or by its ability to bindspecifically to an anti-IL-28 or IL-29 antibody or IL-28 receptor(either soluble or immobilized). The specialized activities of Cysteinemutant IL-28 or IL-29 polypeptides and how to test for them aredisclosed herein. As previously described herein, IL-28 and IL-29polypeptides are characterized by a six-helical-bundle. Thus, thepresent invention further provides fusion proteins encompassing: (a)polypeptide molecules comprising one or more of the helices describedabove; and (b) functional fragments comprising one or more of thesehelices. The other polypeptide portion of the fusion protein may becontributed by another helical-bundle cytokine or interferon, such asIFN-α, or by a non-native and/or an unrelated secretory signal peptidethat facilitates secretion of the fusion protein.

The Cysteine mutant IL-28 or IL-29 polypeptides of the presentinvention, including full-length polypeptides, biologically activefragments, and fusion polypeptides can be produced according toconventional techniques using cells into which have been introduced anexpression vector encoding the polypeptide. As used herein, “cells intowhich have been introduced an expression vector” include both cells thathave been directly manipulated by the introduction of exogenous DNAmolecules and progeny thereof that contain the introduced DNA. Suitablehost cells are those cell types that can be transformed or transfectedwith exogenous DNA and grown in culture, and include bacteria, fungalcells, and cultured higher eukaryotic cells. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocolsin Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

Within another aspect, the present invention provides an expressionvector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a polypeptide asdescribed herein; and a transcription terminator.

The present invention also provides an expression vector comprising anisolated and purified DNA molecule including the following operablylinked elements: a transcription promoter; a DNA segment encoding apolypeptide having at least 90 percent or 95 percent sequence identitywith a polypeptide selected from the group consisting of SEQ ID NOs:19,21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, and 161; and a transcription terminator. TheDNA molecule may further comprise a secretory signal sequence operablylinked to the DNA segment. The encoding polypeptide may further comprisean affinity tag as described herein. The present invention also providesa cultured cell containing the above-described expression vector. Theencoded polypeptide may optionally comprise at least 15, at least 30, atleast 45, or at least 60 sequential amino acids of an amino acidsequence as shown in SEQ ID NOs:19, 21, 23, 25, 27, 29, 36, 37, 38, 39,40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161. Theencoded polypeptide may optionally have antiviral activity, e.g.,hepatitis B and hepatitis C.

Within another aspect the present invention provides a cultured cellcomprising an expression vector as disclosed above.

Within another aspect the present invention provides a method ofproducing a protein comprising: culturing a cell as disclosed aboveunder conditions wherein the DNA segment is expressed; and recoveringthe protein encoded by the DNA segment.

In general, a DNA sequence encoding a Cysteine mutant IL-28 or IL-29polypeptide is operably linked to other genetic elements required forits expression, generally including a transcription promoter andterminator, within an expression vector. The vector will also commonlycontain one or more selectable markers and one or more origins ofreplication, although those skilled in the art will recognize thatwithin certain systems selectable markers may be provided on separatevectors, and replication of the exogenous DNA may be provided byintegration into the host cell genome. Selection of promoters,terminators, selectable markers, vectors and other elements is a matterof routine design within the level of ordinary skill in the art. Manysuch elements are described in the literature and are available throughcommercial suppliers.

To direct a Cysteine mutant IL-28 or IL-29 polypeptide into thesecretory pathway of a host cell, a secretory signal sequence (alsoknown as a leader sequence, prepro sequence or pre sequence) is providedin the expression vector. The secretory signal sequence may be that ofCysteine mutant IL-28 or IL-29, e.g., SEQ ID NO:119 or SEQ ID NO:121, ormay be derived from another secreted protein (e.g., t-PA; see, U.S. Pat.No. 5,641,655) or synthesized de novo. The secretory signal sequence isoperably linked to the Cysteine mutant IL-28 or IL-29 DNA sequence,i.e., the two sequences are joined in the correct reading frame andpositioned to direct the newly synthesized polypeptide into thesecretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain signal sequences may be positioned elsewherein the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

A wide variety of suitable recombinant host cells includes, but is notlimited to, gram-negative prokaryotic host organisms. Suitable strainsof E. coli include W3110, K12-derived strains MM294, TG-1, JM-107, BL21,and UT5600. Other suitable strains include: BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF═, DH5IMCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, ER1647, E. coli K12, E. coli K12 RV308, E. coli K12 C600,E. coli HB101, E. coli K12 C600 R.sub.k-M.sub.k-, E. coli K12 RR1 (see,for example, Brown (ed.), Molecular Biology Labfax (Academic Press1991)). Other gram-negative prokaryotic hosts can include Serratia,Pseudomonas, Caulobacter. Prokaryotic hosts can include gram-positiveorganisms such as Bacillus, for example, B. subtilis and B.thuringienesis, and B. thuringienesis var. israelensis, as well asStreptomyces, for example, S. lividans, S. ambofaciens, S. fradiae, andS. griseofuscus. Suitable strains of Bacillus subtilus include BR151,YB886, MI119, MI120, and B170 (see, for example, Hardy, “BacillusCloning Methods,” in DNA Cloning: A Practical Approach, Glover (ed.)(IRL Press 1985)). Standard techniques for propagating vectors inprokaryotic hosts are well-known to those of skill in the art (see, forexample, Ausubel et al (eds.), Short Protocols in Molecular Biology,3^(rd) Edition (John Wiley & Sons 1995); Wu et al., Methods in GeneBiotechnology (CRC Press, Inc. 1997)). In one embodiment, the methods ofthe present invention use Cysteine mutant IL-28 or IL-29 expressed inthe W3110 strain, which has been deposited at the American Type CultureCollection (ATCC) as ATCC #27325.

When large scale production of Cysteine mutant IL-28 or IL-29 using theexpression system of the present invention is required, batchfermentation can be used. Generally, batch fermentation comprises that afirst stage seed flask is prepared by growing E. coli strains expressingCysteine mutant IL-28 or IL-29 in a suitable medium in shake flaskculture to allow for growth to an optical density (OD) of between 5 and20 at 600 nm. A suitable medium would contain nitrogen from a source(s)such as ammonium sulfate, ammonium phosphate, ammonium chloride, yeastextract, hydrolyzed animal proteins, hydrolyzed plant proteins orhydrolyzed caseins. Phosphate will be supplied from potassium phosphate,ammonium phosphate, phosphoric acid or sodium phosphate. Othercomponents would be magnesium chloride or magnesium sulfate, ferroussulfate or ferrous chloride, and other trace elements. Growth medium canbe supplemented with carbohydrates, such as fructose, glucose,galactose, lactose, and glycerol, to improve growth. Alternatively, afed batch culture is used to generate a high yield of Cysteine mutantIL-28 or IL-29 protein. The Cysteine mutant IL-28 or IL-29 producing E.coli strains are grown under conditions similar to those described forthe first stage vessel used to inoculate a batch fermentation.

Following fermentation the cells are harvested by centrifugation,re-suspended in homogenization buffer and homogenized, for example, inan APV-Gaulin homogenizer (Invensys APV, Tonawanda, N.Y.) or other typeof cell disruption equipment, such as bead mills or sonicators.Alternatively, the cells are taken directly from the fermentor andhomogenized in an APV-Gaulin homogenizer. The washed inclusion body prepcan be solubilized using guanidine hydrochloride (5-8 M) or urea (7-8 M)containing a reducing agent such as beta mercaptoethanol (10-100 mM) ordithiothreitol (5-50 mM). The solutions can be prepared in Tris,phopshate, HEPES or other appropriate buffers. Inclusion bodies can alsobe solubilized with urea (2-4 M) containing sodium lauryl sulfate(0.1-2%). In the process for recovering purified Cysteine mutant IL-28or IL-29 from transformed E. coli host strains in which the Cysteinemutant IL-28 or IL-29 is accumulates as refractile inclusion bodies, thecells are disrupted and the inclusion bodies are recovered bycentrifugation. The inclusion bodies are then solubilized and denaturedin 6 M guanidine hydrochloride containing a reducing agent. The reducedCysteine mutant IL-28 or IL-29 is then oxidized in a controlledrenaturation step. Refolded Cysteine mutant IL-28 or IL-29 can be passedthrough a filter for clarification and removal of insoluble protein. Thesolution is then passed through a filter for clarification and removalof insoluble protein. After the Cysteine mutant IL-28 or IL-29 proteinis refolded and concentrated, the refolded Cysteine mutant IL-28 orIL-29 protein is captured in dilute buffer on a cation exchange columnand purified using hydrophobic interaction chromatography.

Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Manassas, Va. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plantcells, insect cells and avian cells. The use of Agrobacterium rhizogenesas a vector for expressing genes in plant cells has been reviewed bySinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). See, King, L. A. and Possee, R. D., The Baculovirus ExpressionSystem: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. etal., Baculovirus Expression Vectors: A Laboratory Manual, New York,Oxford University Press., 1994; and, Richardson, C. D., Ed., BaculovirusExpression Protocols. Methods in Molecular Biology, Totowa, N.J., HumanaPress, 1995. The second method of making recombinant baculovirusutilizes a transposon-based system described by Luckow (Luckow, V. A, etal., J Virol 67:4566-79, 1993). This system is sold in the Bac-to-Backit (Life Technologies, Rockville, Md.). This system utilizes a transfervector, pFastBac1™ (Life Technologies) containing a Tn7 transposon tomove the DNA encoding the Cysteine mutant IL-28 or IL-29 polypeptideinto a baculovirus genome maintained in E. coli as a large plasmidcalled a “bacmid.” The pFastBac1™ transfer vector utilizes the AcNPVpolyhedrin promoter to drive the expression of the gene of interest, inthis case Cysteine mutant IL-28 or IL-29. However, pFastBac1™ can bemodified to a considerable degree. The polyhedrin promoter can beremoved and substituted with the baculovirus basic protein promoter(also known as Pcor, p6.9 or MP promoter) which is expressed earlier inthe baculovirus infection, and has been shown to be advantageous forexpressing secreted proteins. See, Hill-Perkins, M. S. and Possee, R.D., J. Gen. Virol. 71:971-6, 1990; Bonning, B. C. et al., J. Gen. Virol.75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport, B., J. Biol.Chem. 270:1543-9, 1995. In such transfer vector constructs, a short orlong version of the basic protein promoter can be used. Moreover,transfer vectors can be constructed which replace the native IL-28 orIL-29 secretory signal sequences with secretory signal sequences derivedfrom insect proteins. For example, a secretory signal sequence fromEcdysteroid Glucosyltransferase (EGT), honey bee Meliffin (Invitrogen,Carlsbad, Calif.), or baculovirus gp67 (PharMingen, San Diego, Calif.)can be used in constructs to replace the native IL-28 or IL-29 secretorysignal sequence. In addition, transfer vectors can include an in-framefusion with DNA encoding an epitope tag at the C— or N-terminus of theexpressed Cysteine mutant IL-28 or IL-29 polypeptide, for example, aGlu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci.82:7952-4, 1985). Using techniques known in the art, a transfer vectorcontaining Cysteine mutant IL-28 or IL-29 is transformed into E. Coli,and screened for bacmids which contain an interrupted lacZ geneindicative of recombinant baculovirus. The bacmid DNA containing therecombinant baculovirus genome is isolated, using common techniques, andused to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.Recombinant virus that expresses Cysteine mutant IL-28 or IL-29 issubsequently produced. Recombinant viral stocks are made by methodscommonly used the art.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula poly orpha, Schizosaccharomyces pombe,Ktuyveromyces lactis, Ktuyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533. The use of Pichia methanotica as host for theproduction of recombinant proteins is disclosed in U.S. Pat. Nos.5,955,349, 5,888,768 and 6,001,597, U.S. Pat. No. 5,965,389, U.S. Pat.No. 5,736,383, and U.S. Pat. No. 5,854,039.

It is preferred to purify the polypeptides and proteins of the presentinvention to ≧80% purity, more preferably to ≧90% purity, even morepreferably ≧95% purity, and particularly preferred is a pharmaceuticallypure state, that is greater than 99.9% pure with respect tocontaminating macromolecules, particularly other proteins and nucleicacids, and free of infectious and pyrogenic agents. Preferably, apurified polypeptide or protein is substantially free of otherpolypeptides or proteins, particularly those of animal origin.

Expressed recombinant Cysteine mutant IL-28 or IL-29 proteins (includingchimeric polypeptides and multimeric proteins) are purified byconventional protein purification methods, typically by a combination ofchromatographic techniques. See, in general, Affinity Chromatoiraphy:Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden,1988; and Scopes, Protein Purification: Principles and Practice,Springer-Verlag, New York, 1994. Proteins comprising a polyhistidineaffinity tag (typically about 6 histidine residues) are purified byaffinity chromatography on a nickel chelate resin. See, for example,Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteins comprising aglu-glu tag can be purified by immunoaffinity chromatography accordingto conventional procedures. See, for example, Grussenmeyer et al.,supra. Maltose binding protein fusions are purified on an amylose columnaccording to methods known in the art.

Cysteine mutant IL-28 or IL-29 polypeptides can also be prepared throughchemical synthesis according to methods known in the art, includingexclusive solid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. See, for example,Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid PhasePeptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, Ill.,1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al.,Solid Phase Pelptide Synthesis: A Practical Approach, IRL Press, Oxford,1989. In vitro synthesis is particularly advantageous for thepreparation of smaller polypeptides.

Using methods known in the art, Cysteine mutant IL-28 or IL-29 proteinscan be prepared as monomers or multimers; glycosylated ornon-glycosylated; pegylated or non-pegylated; fusion proteins; and mayor may not include an initial methionine amino acid residue. Cysteinemutant IL-28 or IL-29 conjugates used for therapy may comprisepharmaceutically acceptable water-soluble polymer moieties. Conjugationof interferons with water-soluble polymers has been shown to enhance thecirculating half-life of the interferon, and to reduce theimmunogenicity of the polypeptide (see, for example, Nieforth et al.,Clin. Pharmacol. Ther. 59:636 (1996), and Monkarsh et al, Anal. Biochem.247:434 (1997)).

Suitable water-soluble polymers include polyethylene glycol (PEG),monomethoxy-PEG, mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinylpyrrolidone)PEG, tresyl monomethoxy PEG, monomethoxy-PEGpropionaldehyde, PEG propionaldehyde, bis-succinimidyl carbonate PEG,propylene glycol homopolymers, a polypropylene oxide/ethylene oxideco-polymer, polyoxyethylated polyols (e.g., glycerol), monomethoxy-PEGbutyraldehyde, PEG butyraldehyde, monomethoxy-PEG acetaldehyde, PEGacetaldehyde, methoxyl PEG-succinimidyl propionate, methoxylPEG-succinimidyl butanoate, polyvinyl alcohol, dextran, cellulose, orother carbohydrate-based polymers. Suitable PEG may have a molecularweight from about 600 to about 60,000, including, for example, 5,000daltons, 12,000 daltons, 20,000 daltons, 30,000 daltons, and 40,000daltons, which can be linear or branched. A Cysteine mutant IL-28 orIL-29 conjugate can also comprise a mixture of such water-solublepolymers.

One example of a Cysteine mutant IL-28 or IL-29 conjugate comprises aCysteine mutant IL-28 or IL-29 moiety and a polyalkyl oxide moietyattached to the N-terminus of the Cysteine mutant IL-28 or IL-29 moiety.PEG is one suitable polyalkyl oxide. As an illustration, Cysteine mutantIL-28 or IL-29 can be modified with PEG, a process known as“PEGylation.” PEGylation of Cysteine mutant IL-28 or IL-29 can becarried out by any of the PEGylation reactions known in the art (see,for example, EP 0 154 316, Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems 9:249 (1992), Duncan and Spreafico,Clin. Pharmacokinet. 27:290 (1994), and Francis et al., Int J Hematol68:1 (1998)). For example, PEGylation can be performed by an acylationreaction or by an alkylation reaction with a reactive polyethyleneglycol molecule. In an alternative approach, Cysteine mutant IL-28 orIL-29 conjugates are formed by condensing activated PEG, in which aterminal hydroxy or amino group of PEG has been replaced by an activatedlinker (see, for example, Karasiewicz et at., U.S. Pat. No. 5,382,657).

PEGylation by acylation typically requires reacting an active esterderivative of PEG with a Cysteine mutant IL-28 or IL-29 polypeptide. Anexample of an activated PEG ester is PEG esterified toN-hydroxysuccinimide. As used herein, the term “acylation” includes thefollowing types of linkages between Cysteine mutant IL-28 or IL-29 and awater-soluble polymer: amide, carbamate, urethane, and the like. Methodsfor preparing PEGylated Cysteine mutant IL-28 or IL-29 by acylation willtypically comprise the steps of (a) reacting an Cysteine mutant IL-28 orIL-29 polypeptide with PEG (such as a reactive ester of an aldehydederivative of PEG) under conditions whereby one or more PEG groupsattach to Cysteine mutant IL-28 or IL-29, and (b) obtaining the reactionproduct(s). Generally, the optimal reaction conditions for acylationreactions will be determined based upon known parameters and desiredresults. For example, the larger the ratio of PEG: Cysteine mutant IL-28or IL-29, the greater the percentage of polyPEGylated Cysteine mutantIL-28 or IL-29 product.

PEGylation by alkylation generally involves reacting a terminalaldehyde, e.g., propionaldehyde, butyraldehyde, acetaldehyde, and thelike, derivative of PEG with Cysteine mutant IL-28 or IL-29 in thepresence of a reducing agent. PEG groups are preferably attached to thepolypeptide via a —CH₂—NH₂ group.

Derivatization via reductive alkylation to produce a monoPEGylatedproduct takes advantage of the differential reactivity of differenttypes of primary amino groups available for derivatization. Typically,the reaction is performed at a pH that allows one to take advantage ofthe pKa differences between the ε-amino groups of the lysine residuesand the α-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water-soluble polymer thatcontains a reactive group such as an aldehyde, to a protein iscontrolled. The conjugation with the polymer occurs predominantly at theN-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups.

Reductive alkylation to produce a substantially homogenous population ofmonopolymer Cysteine mutant IL-28 or IL-29 conjugate molecule cancomprise the steps of: (a) reacting a Cysteine mutant IL-28 or IL-29polypeptide with a reactive PEG under reductive alkylation conditions ata pH suitable to permit selective modification of the α-amino group atthe amino terminus of the Cysteine mutant IL-28 or IL-29, and (b)obtaining the reaction product(s). The reducing agent used for reductivealkylation should be stable in aqueous solution and preferably be ableto reduce only the Schiff base formed in the initial process ofreductive alkylation. Preferred reducing agents include sodiumborohydride, sodium cyanoborohydride, dimethylamine borane,trimethylamine borane, and pyridine borane.

For a substantially homogenous population of monopolymer Cysteine mutantIL-28 or IL-29 conjugates, the reductive alkylation reaction conditionsare those that permit the selective attachment of the water-solublepolymer moiety to the N-terminus of Cysteine mutant IL-28 or IL-29. Suchreaction conditions generally provide for pKa differences between thelysine amino groups and the α-amino group at the N-terminus. The pH alsoaffects the ratio of polymer to protein to be used. In general, if thepH is lower, a larger excess of polymer to protein will be desiredbecause the less reactive the N-terminal α-group, the more polymer isneeded to achieve optimal conditions. If the pH is higher, the polymer:Cysteine mutant IL-28 or IL-29 need not be as large because morereactive groups are available. Typically, the pH will fall within therange of 3-9, or 3-6. Another factor to consider is the molecular weightof the water-soluble polymer. Generally, the higher the molecular weightof the polymer, the fewer number of polymer molecules which may beattached to the protein. For PEGylation reactions, the typical molecularweight is about 2 kDa to about 100 kDa, about 5 kDa to about 50 kDa,about 12 kDa to about 40 kDa, or about 20 kDa to about 30 kDa. The molarratio of water-soluble polymer to Cysteine mutant IL-28 or IL-29 willgenerally be in the range of 1:1 to 100:1. Typically, the molar ratio ofwater-soluble polymer to Cysteine mutant IL-28 or IL-29 will be 1:1 to20:1 for polyPEGylation, and 1:1 to 5:1 for monoPEGylation.

General methods for producing conjugates comprising interferon andwater-soluble polymer moieties are known in the art. See, for example,Karasiewicz et at., U.S. Pat. No. 5,382,657, Greenwald et at., U.S. Pat.No. 5,738, 846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996),Monkarsh et al., Anal. Biochem. 247:434 (1997). PEGylated species can beseparated from unconjugated Cysteine mutant IL-28 or IL-29 polypeptidesusing standard purification methods, such as dialysis, ultrafiltration,ion exchange chromatography, affinity chromatography, size exclusionchromatography, and the like.

The Cysteine mutant IL-28 or IL-29 molecules of the present inventionare capable of specifically binding the IL-28 receptor and/or acting asan antiviral agent. The binding of Cysteine mutant IL-28 or 11-29polypeptides to the IL-28 receptor can be assayed using establishedapproaches. Cysteine mutant IL-28 or IL-29 can be iodinated using aniodobead (Pierce, Rockford, Ill.) according to manufacturer'sdirections, and the ¹²⁵I-IL-28 or ¹²⁵I-IL-29 can then be used asdescribed below.

In a first approach fifty nanograms of ¹²⁵I-IL-28 or ¹²⁵I-IL-29 can becombind with 1000 ng of IL-28 receptor human IgG fusion protein, in thepresence or absence of possible binding competitors including unlabeledcysteine mutant IL-28, cysteine mutant IL-29, IL-28, or IL-29. The samebinding reactions would also be performed substituting other cytokinereceptor human IgG fusions as controls for specificity. Followingincubation at 4° C., protein-G (Zymed, San Fransisco, Calif.) is addedto the reaction, to capture the receptor-IgG fusions and any proteinsbound to them, and the reactions are incubated another hour at 4° C. Theprotein-G sepharose is then collected, washed three times with PBS and¹²⁵I-IL-28 or ¹²⁵I-IL-29 bound is measure by gamma counter (PackardInstruments, Downers Grove, Ill.).

In a second approach, the ability of molecules to inhibit the binding of¹²⁵I-IL-28 or ¹²¹I-IL-29 to plate bound receptors can be assayed. Afragment of the IL-28 receptor, representing the extracellular, ligandbinding domain, can be adsorbed to the wells of a 96 well plate byincubating 100 μl of 1 g/mL solution of receptor in the plate overnight.In a second form, a receptor-human IgG fusion can be bound to the wellsof a 96 well plate that has been coated with an antibody directedagainst the human IgG portion of the fusion protein. Following coatingof the plate with receptor the plate is washed, blocked with SUPERBLOCK(Pierce, Rockford, Ill.) and washed again. Solutions containing a fixedconcentration of ¹²⁵I-IL-28 or ¹²⁵I-IL-29 with or without increasingconcentrations of potential binding competitors including, Cysteinmutant IL-28, cysteine mutant IL-29, IL-28 and IL-29, and 100 μl of thesolution added to appropriate wells of the plate. Following a one hourincubation at 4° C. the plate is washed and the amount ¹²⁵I-IL-28 or¹²⁵I-IL-29 bound determined by counting (Topcount, Packard Instruments,Downers grove, Ill.). The specificity of binding of ¹²⁵I-IL-28 or¹²⁵I-IL-29 can be defined by receptor molecules used in these bindingassays as well as by the molecules used as inhibitors.

In addition to pegylation, human albumin can be genetically coupled to apolypeptide of the present invention to prolong its half-life. Humanalbumin is the most prevalent naturally occurring blood protein in thehuman circulatory system, persisting in circulation in the body for overtwenty days. Research has shown that therapeutic proteins geneticallyfused to human albumin have longer half-lives. An IL28 or IL29 albuminfusion protein, like pegylation, may provide patients with long-actingtreatment options that offer a more convenient administration schedule,with similar or improved efficacy and safety compared to currentlyavailable treatments (U.S. Pat. No. 6,165,470; Syed et al., Blood,89(9):3243-3253 (1997); Yeh et al., Proc. Natl. Acad. Sci. USA,89:1904-1908 (1992); and Zeisel et al., Horm. Res., 37:5-13 (1992)).

Like the aforementioned peglyation and human albumin, an Fc portion ofthe human IgG molecule can be fused to a polypeptide of the presentinvention. The resultant fusion protein may have an increasedcirculating half-life due to the Fc moiety (U.S. Pat. No. 5,750,375,U.S. Pat. No. 5843,725, U.S. Pat. No. 6,291,646; Barouch et al., Journalof Immunology, 61:1875-1882 (1998); Barouch et al., Proc. Natl. Acad.Sci. USA, 97(8):4192-4197 (Apr. 11, 2000); and Kim et al., TransplantProc., 30(8):4031-4036 (December 1998)).

Methods for detection and diagnosis of viral infections are well knownto those skilled in the art. The exact method used for measuring areduction in virus in response to administration of molecules of thepresent invention will be dependent upon the species of virus andwhether the infection is in vitro or in vivo. If the infection is invivo, measurement and detection of infection and changes in the levelsof infection, can vary depending on subject infected, type of viralinfection, and the like. For example, methods include, but are notlimited to, measuring changes in CD4 cell counts, serologic tests,measuring the DNA of the virus and RNA of the virus by conventional andreal-time quantitative polymerase chain reaction assays, viral inducedantibody levels, immunofluorescence and enzyme-linked immunosorbantassays, cytopathic effects, and histology.

Antiviral effects may be direct or indirect. An example of a directantiviral effect is, for example, where Cysteine mutant IL-28 or IL-29polypeptide competes for a viral receptor or co-receptor to block viralinfection. Cysteine mutant IL-28 or IL-29 may be given parentally toprevent viral infection or to reduce ongoing viral replication andre-infection (Gayowski, T. et al., Transplantation 64:422-426, 1997). Anexample of an indirect antiviral effect is, for example, where aCysteine mutant IL-28 or IL-29 may bind CD4 or another leukocytereceptor and exhibit antiviral effects by modulating the effects of theimmune response.

Of particular interest is the use of Cysteine mutant IL-28 or IL-29 asan antiviral therapeutic for viral leukemias (HTLV), AIDS (HIV), orgastrointestinal viral infections caused by, for example, rotavirus,calicivirus (e.g., Norwalk Agent) and certain strains of pathogenicadenovirus, Hepatitis B and C.

Additional types of viral infections for Cysteine mutant IL-28 or IL-29use include, but are not limited to: infections caused by DNA Viruses(e.g., Herpes Viruses such as Herpes Simplex viruses, Epstein-Barrvirus, Cytomegalovirus; Pox viruses such as Variola (small pox) virus;Hepadnaviruses (e.g, Hepatitis B virus); Papilloma viruses;Adenoviruses); RNA Viruses (e.g., HIV I, II; HTLV I, II; Poliovirus;Hepatitis A; coronoviruses, such as sudden acute respiratory syndrome(SARS); Orthomyxoviruses (e.g., Influenza viruses); Paramyxoviruses(e.g., Measles virus); Rabies virus; Hepatitis C virus), Flaviviruses,Influenza viruses; caliciviruses; rabies viruses, rinderpest viruses,Arena virus, and the like. Moreover, examples of the types ofvirus-related diseases for which Cysteine mutant IL-28 or IL-29 could beused include, but are not limited to: Acquired immunodeficiency; SevereAcute Respiratory Syndrome (SARS); Hepatitis; Gastroenteritis;Hemorrhagic diseases; Enteritis; Carditis; Encephalitis; Paralysis;Brochiolitis; Upper and lower respiratory disease; RespiratoryPapillomatosis; Arthritis; Disseminated disease, Meningitis,Mononucleosis. In addition, Cysteine mutant IL-28 or IL-29 can be usedin various applications for antiviral immunotherapy, and in conjunctionwith other cytokines, other protein or small molecule antivirals, andthe like.

Clinically, diagnostic tests for HCV include serologic assays forantibodies and molecular tests for viral particles. Enzyme immunoassaysare available (Vrielink et al., Transfusion 37:845-849, 1997), but mayrequire confirmation using additional tests such as an immunoblot assay(Pawlotsky et al., Hepatology 27:1700-1702, 1998). Qualitative andquantitative assays generally use polymerase chain reaction techniques,and are preferred for assessing viremia and treatment response (Poynardet al., Lancet 352:1426-1432, 1998; McHutchinson et al., N. Engl. J.Med. 339:1485-1492, 1998). Several commercial tests are available, suchas, quantitative RT-PCR (Amplicor HCV Monitor™, Roche Molecular Systems,Branchburg, N.J.) and a branched DNA (deoxyribonucleic acid) signalamplification assay (Quantiplex™ HCV RNA Assay [bDNA], Chiron Corp.,Emeryville, Calif.). A non-specific laboratory test for HCV infectionmeasures alanine aminotransferase level (ALT) and is inexpensive andreadily available (National Institutes of Health Consensus DevelopmentConference Panel, Hepatology 26 (Suppl. 1):2S-10S, 1997). Histologicevaluation of liver biopsy is generally considered the most accuratemeans for determining HCV progression (Yano et al., Hepatology23:1334-1340, 1996.) For a review of clinical tests for HCV, see, Laueret al., N. Engl. J. Med. 345:41-52, 2001.

There are several in vivo models for testing HBV and HCV that are knownto those skilled in art. With respect to HCV, for example, the HCVReplicon model is a cell-based system to study the effectiveness of adrug to inhibit HCV replication (Blight et al., Science,290(5498):1972-1974 (Dec. 8, 2000); and Lohmann et al., Science,285(5424):110-113 (Jul. 2, 1999)). A well-known and accepted in vitroHBV model to one of skill in the art can be used to determine theanti-HBV activity of a test molecule is disclosed in Korba et al.,Antiviral Res., 19(1):55-70 (1992) and Korba et al., Antiviral Res.,15(3):217-228 (1991).

For example, the effects of Cysteine mutant IL-28 or IL-29 on mammalsinfected with HBV can accessed using a woodchuck model. Briefly,woodchucks chronically infected with woodchuck hepatitis virus (WHV)develop hepatitis and hepatocellular carcinoma that is similar todisease in humans chronically infected with HBV. The model has been usedfor the preclinical assessment of antiviral activity. A chronicallyinfected WHV strain has been established and neonates are inoculatedwith serum to provide animals for studying the effects of certaincompounds using this model. (For a review, see, Tannant et al., ILAR J.42 (2):89-102, 2001). Chimpanzees may also be used to evaluate theeffect of Cysteine mutant IL-28 or IL-29 on HBV infected mammals. Usingchimpanzees, characterization of HBV was made and these studiesdemonstrated that the chimpanzee disease was remarkably similar to thedisease in humans (Barker et al., J. Infect. Dis. 132:451-458, 1975 andTabor et al., J. Infect. Dis. 147:531-534, 1983.) The chimpanzee modelhas been used in evaluating vaccines (Prince et al., In: Vaccines 97Cold Spring Harbor Laboratory Press, 1997.) Therapies for HIV areroutinely tested using non-human primates infected with simianimmunodeficiency viruses (for a review, see, Hirsch et al., Adv.Pharmcol. 49:437-477, 2000 and Nathanson et al., AIDS 13 (suppl.A):S113-S120, 1999.) For a review of use of non-human primates in HIV,hepatitis, malaria, respiratory syncytial virus, and other diseases,see, Sibal et al., ILAR J. 42 (2):74-84, 2001. A recently developedtransgenic mouse model (Guidotti et al., Journal of Virology69:6158-6169, 1995) supports the replication of high levels ofinfectious HBV and has been used as a chemotherapeutic model for HBVinfection. Transgenic mice are treated with antiviral drugs and thelevels of HBV DNA and RNA are measured in the transgenic mouse liver andserum following treatment. HBV protein levels can also be measured inthe transgenic mouse serum following treatment. This model has been usedto evaluate the effectiveness of lamivudine and IFN-alpha in reducingHBV viral titers (Morrey et al., Antiviral Therapy 3 :59-68, 1998).

Moreover, Cysteine mutant IL-28 or IL-29 polyeptides and proteins of thepresent invention can be characterized by their activity, that is,modulation of the proliferation, differentiation, migration, adhesion,gene expression or metabolism of responsive cell types. Biologicalactivity of Cysteine mutant IL-28 or IL-29 polypeptides and proteins isassayed using in vitro or in vivo assays designed to detect cellproliferation, differentiation, migration or adhesion; or changes ingene expression or cellular metabolism (e.g., production of other growthfactors or other macromolecules). Many suitable assays are known in theart, and representative assays are disclosed herein. Assays usingcultured cells are most convenient for screening, such as fordetermining the effects of amino acid substitutions, deletions, orinsertions.

Activity of Cysteine mutant IL-28 or IL-29 proteins can be measured invitro using cultured cells or in vivo by administering molecules of theclaimed invention to an appropriate animal model. Assays measuring cellproliferation or differentiation are well known in the art. For example,assays measuring proliferation include such assays as chemosensitivityto neutral red dye (Cavanaugh et al., Investigational New Drugs8:347-354, 1990), incorporation of radiolabelled nucleotides (asdisclosed by, e.g., Raines and Ross, Methods Enzmol. 109:749-773, 1985;Wahl et al., Mol. Cell Biol. 8:5016-5025, 1988; and Cook et al.,Analytical Biochem. 179:1-7, 1989), incorporation of5-bromo-2′-deoxyuridine (BrdU) in the DNA of proliferating cells(Porstmann et al., J. Immunol. Methods 82:169-179, 1985), and use oftetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley etal., Cancer Res. 48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84,1995; and Scudiero et al., Cancer Res. 48:4827-4833, 1988).Differentiation can be assayed using suitable precursor cells that canbe induced to differentiate into a more mature phenotype. Assaysmeasuring differentiation include, for example, measuring cell-surfacemarkers associated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Watt, FASEB,5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv.Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporatedherein by reference).

Cysteine mutant IL-28 or IL-29 polypeptide activity may also be detectedusing assays designed to measure IL-28- and IL-29-induced production ofone or more additional growth factors or other macromolecules. Certainmembers of the protein family comprising IL-28 and IL-29 have been shownto increase circulating monocyte numbers in vivo. Monocyte activation isimportant in both innate and adaptive immunity. For example, activationof monocytes has been shown to stimulate antigen presentation by severalmechanisms. Antigen presentation promotes activation and proliferationof T-cells, both cytotoxic and helper T cells. The maturation andactivation of dendritic cells also promotes activation of T cells andboth innate and adaptive immunity. Increases in activated monocytes andmacrophages have also been shown to increase cytolytic activity.Therefore, Cysteine mutant IL-28 or IL-29 will be useful as ananti-infectious agent, enhancing innate, cell-mediated and humoralimmune responses. Increases in ICAM staining in CD 14+ monocytes wasseen suggesting that IL-28 and IL-29 play a role in monocyte activation.While data show that family members promote an anti-viral response tovirus, bacteria and parasites may also be affected.

Monocyte activation assays are carried out (1) to look for the abilityof Cysteine mutant IL-28 or IL-29 proteins to further stimulate monocyteactivation, and (2) to examine the ability of Cysteine mutant IL-28 orIL-29 proteins to modulate attachment-induced or endotoxin-inducedmonocyte activation (Fuhlbrigge et al., J. Immunol. 138: 3799-3802,1987). IL-1α and TNFα levels produced in response to activation aremeasured by ELISA (Biosource, Inc. Camarillo, Calif.).Monocyte/macrophage cells, by virtue of CD14 (LPS receptor), areexquisitely sensitive to endotoxin, and proteins with moderate levels ofendotoxin-like activity will activate these cells.

Increased levels of monocytes suggest that Cysteine mutant IL-28 orIL-29 may have a direct effect on myeloid progenitor cells in the bonemarrow. Increasing differentiation of myeloid progenitor cells tomonocytes is essential in restoring immnunocompetency, for example,after chemotherapy. Thus, administration of Cysteine mutant IL-28 orIL-29 to patients receiving chemotherapy could promote their recoveryand ability to resist infection commonly associated with chemotherapyregimens. Thus, methods for expanding the numbers of monocytes ormonocyte progenitor cells by either culturing bone marrow or peripheralblood cells with the molecules of the present invention such that thereis an increase in the monocyte or monocyte progenitor cells forachieving this effect in vitro or ex vivo. The present invention alsoprovides for the in vivo administration of the molecules of the presentinvention to a mammal needing increased monocyte or monocyte progenitorcells. Increased monocyte and monocyte progenitor cells can be measuredusing methods well known to clinicians, physicians, and other personsskilled the art. Monocyte cells are included in the myeloid lineage ofhematopoietic cells, so affects on other cells in that lineage would notbe unusual. For example, when a factor facilitates the differentiationor proliferation of one type of cell in the myeloid or lymphoid lineage,this can affect production of other cells with a common progenitor orstem cell.

Hematopoietic activity of Cysteine mutant IL-28 or IL-29 proteins can beassayed on various hematopoietic cells in culture. Preferred assaysinclude primary bone marrow colony assays and later stagelineage-restricted colony assays, which are known in the art (e.g.,Holly et al., WIPO Publication WO 95/21920). Marrow cells plated on asuitable semi-solid medium (e.g., 50% methylcellulose containing 15%fetal bovine serum, 10% bovine serum albumin, and 0.6% PSN antibioticmix) are incubated in the presence of test polypeptide, then examinedmicroscopically for colony formation. Known hematopoietic factors areused as controls. Mitogenic activity of Cysteine mutant IL-28 or IL-29polypeptides on hematopoietic cell lines can be measured as disclosedabove.

Cell migration is assayed essentially as disclosed by Kahler et al.(Arteriosclerosis Thrombosis and Vascular Biology 17:932-939, 1997). Aprotein is considered to be chemotactic if it induces migration of cellsfrom an area of low protein concentration to an area of high proteinconcentration. A typical assay is performed using modified Boydenchambers with a polystryrene membrane separating the two chambers(Transwell; Coming Costar Corp.). The test sample, diluted in mediumcontaining 1% BSA, is added to the lower chamber of a 24-well platecontaining Transwells. Cells are then placed on the Transwell insertthat has been pretreated with 0.2% gelatin. Cell migration is measuredafter 4 hours of incubation at 37° C. Non-migrating cells are wiped offthe top of the Transwell membrane, and cells attached to the lower faceof the membrane are fixed and stained with 0.1% crystal violet. Stainedcells are then extracted with 10% acetic acid and absorbance is measuredat 600 nm. Migration is then calculated from a standard calibrationcurve. Cell migration can also be measured using the matrigel method ofGrant et al. (“Angiogenesis as a component of epithelial-mesenchymalinteractions” in Goldberg and Rosen, Epithelial-Mesenchymal Interactionin Cancer, Birkhäuser Verlag, 1995, 235-248; Baatout, AnticancerResearch 17:451-456, 1997).

Cell adhesion activity is assayed essentially as disclosed by LaFleur etal. (J. Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter platesare coated with the test protein, non-specific sites are blocked withBSA, and cells (such as smooth muscle cells, leukocytes, or endothelialcells) are plated at a density of approximately 10⁴-10⁵ cells/well. Thewells are incubated at 37° C. (typically for about 60 minutes), thennon-adherent cells are removed by gentle washing. Adhered cells arequantitated by conventional methods (e.g., by staining with crystalviolet, lysing the cells, and determining the optical density of thelysate). Control wells are coated with a known adhesive protein, such asfibronectin or vitronectin.

Expression of Cysteine mutant IL-28 or IL-29 polynucleotides in animalsprovides models for further study of the biological effects ofoverproduction or inhibition of protein activity in vivo. IL-28- orIL-29-encoding polynucleotides and antisense polynucleotides can beintroduced into test animals, such as mice, using viral vectors or nakedDNA, or transgenic animals can be produced.

One in vivo approach for assaying proteins of the present inventionutilizes viral delivery systems. Exemplary viruses for this purposeinclude adenovirus, herpesvirus, retroviruses, vaccinia virus, andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acids. For review, see Becker et al., Meth. CellBiol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine4:44-53, 1997. The adenovirus system offers several advantages.Adenovirus can (i) accommodate relatively large DNA inserts; (ii) begrown to high-titer; (iii) infect a broad range of mammalian cell types;and (iv) be used with many different promoters including ubiquitous,tissue specific, and regulatable promoters. Because adenoviruses arestable in the bloodstream, they can be administered by intravenousinjection. Also see, Wu et al., J. Biol. Chem. 263:14621-14624, 1988; Wuet al., J. Biol. Chem. 267:963-967, 1992; and Johnston and Tang, Meth.Cell Biol. 43:353-365, 1994.

Transgenic mice, engineered to express a Cysteine mutant IL-28 or IL-29gene, and mice that exhibit a complete absence of Cysteine mutant IL-28or IL-29 gene function, referred to as “knockout mice” (Snouwaert etal., Science 257:1083, 1992), can also be generated (Lowell et al.,Nature 366:740-742, 1993). These mice can be employed to study theCysteine mutant IL-28 or IL-29 gene and the protein encoded thereby inan in vivo system. Preferred promoters for transgenic expression includepromoters from metallothionein and albumin genes.

Most cytokines as well as other proteins produced by activatedlymphocytes play an important biological role in cell differentiation,activation, recruitment and homeostasis of cells throughout the body.Cysteine mutant IL-28 or IL-29 and inhibitors of their activity areexpected to have a variety of therapeutic applications. Thesetherapeutic applications include treatment of diseases which requireimmune regulation, including autoimmune diseases such as rheumatoidarthritis, multiple sclerosis, myasthenia gravis, systemic lupuserythematosis, and diabetes. IL-28 or IL-29 may be important in theregulation of inflammation, and therefore would be useful in treatingrheumatoid arthritis, asthma and sepsis. There may be a role of IL-28 orIL-29 in mediating tumorgenesis, whereby a Cysteine mutant IL-28 orIL-29 antagonist would be useful in the treatment of cancer. IL-28 orIL-29 may be useful in modulating the immune system, whereby Cysteinemutant IL-28 or IL-29 antagonists may be used for reducing graftrejection, preventing graft-vs-host disease, boosting immunity toinfectious diseases, treating immunocompromised patients (e.g., HIV¹patients), or in improving vaccines.

Members of the protein family of the present invention have been shownto have an antiviral effect that is similar to interferon-α. Interferonhas been approved in the United States for treatment of autoimmunediseases, condyloma acuminatum, chronic hepatitis C, bladder carcinoma,cervical carcinoma, laryngeal papillomatosis, fungoides mycosis, chronichepatitis B, Kaposi's sarcoma in patients infected with humanimmunodeficiency virus, malignant melanoma, hairy cell leukemia, andmultiple sclerosis. In addition, Cysteine mutant IL-28 or IL-29 may beused to treat forms of arteriosclerosis, such as atherosclerosis, byinhibiting cell proliferation. Accordingly, the present inventioncontemplates the use of Cysteine mutant IL-28 or IL-29 proteins,polypeptides, and peptides having IL-28 and IL-29 activity to treat suchconditions, as well as to treat retinopathy. The present invention alsocontemplates the use of Cysteine mutant IL-28 or IL-29 proteins,polypeptides, and peptides having IL-28 and IL-29 activity to treatlymphoproliferative disorders, including B-cell lymphomas, chroniclymphocytic leukemia, acute lymphocytic leukemia, Non-Hodkin'slymphomas, multiple myeloma, acute myelocytic leukemia, chronicmyelocytic leukemia.

Interferons have also been shown to induce the expression of antigens bycultured cells (see, for example, Auth et al., Helpatoloiy 18:546(1993), Guadagni et al., Int. J. Biol. Markers 9:53 (1994), Girolomoniet al., Eur. J. Immunol. 25:2163 (1995), and Maciejewski et al., Blood85:3183 (1995). This activity enhances the ability to identify new tumorassociated antigens in vitro. Moreover, the ability of interferons toaugment the level of expression of human tumor antigens indicates thatinterferons can be useful in an adjuvant setting for immunotherapy orenhance immunoscintigraphy using anti-tumor antigen antibodies (Guadagniet al., Cancer Immunol. Immunother. 26:222 (1988); Guadagni et al., Int.J. Biol. Markers 9:53 (1994)). Thus, the present invention includes theuse of Cysteine mutant IL-28 or IL-29 proteins, polypeptides andpeptides having IL-28 and IL-29 activity as an adjuvant forimmunotherapy or to improve immunoscintigraphy using anti-tumor antigenantibodies.

The activity and effect of Cysteine mutant IL-28 or IL-29 on tumorprogression and metastasis can be measured in vivo. Several syngeneicmouse models have been developed to study the influence of polypeptides,compounds or other treatments on tumor progression. In these models,tumor cells passaged in culture are implanted into mice of the samestrain as the tumor donor. The cells will develop into tumors havingsimilar characteristics in the recipient mice, and metastasis will alsooccur in some of the models. Appropriate tumor models for our studiesinclude the Lewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma(ATCC No. CRL-6323), amongst others. These are both commonly used tumorlines, syngeneic to the C57BL6 mouse, that are readily cultured andmanipulated in vitro. Tumors resulting from implantation of either ofthese cell lines are capable of metastasis to the lung in C57BL6 mice.The Lewis lung carcinoma model has recently been used in mice toidentify an inhibitor of angiogenesis (O'Reilly M S, et al. Cell 79:315-328,1994). C57BL6/J mice are treated with an experimental agenteither through daily injection of recombinant protein, agonist orantagonist or a one-time injection of recombinant adenovirus. Three daysfollowing this treatment, 10⁵ to 10⁶ cells are implanted under thedorsal skin. Alternatively, the cells themselves may be infected withrecombinant adenovirus, such as one expressing Cysteine mutant IL-28 andIL-29, before implantation so that the protein is synthesized at thetumor site or intracellularly, rather than systemically. The micenormally develop visible tumors within 5 days. The tumors are allowed togrow for a period of up to 3 weeks, during which time they may reach asize of 1500-1800 mm³ in the control treated group. Tumor size and bodyweight are carefully monitored throughout the experiment. At the time ofsacrifice, the tumor is removed and weighed along with the lungs and theliver. The lung weight has been shown to correlate well with metastatictumor burden. As an additional measure, lung surface metastases arecounted. The resected tumor, lungs and liver are prepared forhistopathological examination, immunohistochemistry, and in situhybridization, using methods known in the art and described herein. Theinfluence of the expressed polypeptide in question, e.g., Cysteinemutant IL-28 and IL-29, on the ability of the tumor to recruitvasculature and undergo metastasis can thus be assessed. In addition,aside from using adenovirus, the implanted cells can be transientlytransfected with Cysteine mutant IL-28 and IL-29. Use of stable Cysteinemutant IL-28 or IL-29 transfectants as well as use of induceablepromoters to activate Cysteine mutant IL-28 or IL-29 expression in vivoare known in the art and can be used in this system to assess inductionof metastasis. Moreover, purified Cysteine mutant IL-28 or IL-29conditioned media can be directly injected in to this mouse model, andhence be used in this system. For general reference see, O'Reilly M S,et al. Cell 79:315-328, 1994; and Rusciano D, et al. Murine Models ofLiver Metastasis. Invasion Metastasis 14:349-361, 1995.

Cysteine mutant IL-28 or IL-29 can also be used to treat myocarditis, adisorder that arises when the heart is involved in an inflammatoryprocess. The infiltration of lymphocytes and myocytolysis is thought toresult after infection by virus, bacteria, fungi or parasites (see, forexample, Brodison et al., J. Infection 37:99 (1998)). Cysteine mutantIL-28 or IL-29 can be injected intravenously or subcutaneously to treatinfections associated with myocarditis. Cysteine mutant IL-28 or IL-29can also be administered intravenously as an immunoregulatory cytokinein the treatment of autoimmune myocarditis. Interferon dosages can beextrapolated using a autoimmune model of myocarditis in the A/J mouse(Donermeyer, et al., J. Exp. Med. 182:1291 (1995)).

Recent reports have highlighted the role of type I interferons in theprevention of viral-induced diabetes by inducing a strong antiviralstate in pancreatic beta cells early during viral infection (Flodstroemet al., Nature Immunology 3, 373-382 (2002)). This prevents the loss ofbeta cells due to viral-induced cell death and autoimmunity thataccompanies it. Cysteine mutant IL-28 or IL-29 also induce an antiviralstate in cells that express the IL-28 receptor. IL-28 receptor is highlyexpressed in pancreatic tissue and therefore IL-28 and IL-29 may play arole in prevention of viral-induced diabetes due to beta cell death. Inaddition, the role of type I interferons in prevention of viral-induceddiabetes may be extended to other viral-induced autoimmune diseases andtherefore, IL-28 and IL-29 may also play a role in prevention of otherdiseases such as muscular sclerosis, lupus, and viral-induced autoimmunediseases in tissues that express the IL-28 receptor.

Cysteine mutant IL-28 or IL-29 polypeptides can be administered alone orin combination with other vasculogenic or angiogenic agents, includingVEGF. When using Cysteine mutant IL-28 or IL-29 in combination with anadditional agent, the two compounds can be administered simultaneouslyor sequentially as appropriate for the specific condition being treated.

Cysteine mutant IL-28 or IL-29 will be useful in treating tumorgenesis,and therefore would be useful in the treatment of cancer. An IL-28 mayinhibit B-cell tumor lines suggesting that there may be therapeuticbenefit in treating patients with Cysteine mutant IL-28 or IL-29 inorder to induce the B cell tumor cells into a less proliferative state.The ligand could be administered in combination with other agentsalready in use including both conventional chemotherapeutic agents aswell as immune modulators such as interferon alpha. Alpha/betainterferons have been shown to be effective in treating some leukemiasand animal disease models, and the growth inhibitory effects ofinterferon-alpha and Cysteine mutant IL-28 or IL-29 may be additive forB-cell tumor-derived cell lines.

Within another aspect, the present invention provides a pharmaceuticalformulation comprising an isolated polypeptide selected from the groupconsisting of SEQ ID NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27,29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, and 161, and a pharmaceutically acceptable vehicle.

For pharmaceutical use, Cysteine mutant IL-28 or IL-29 proteins areformulated for topical or parenteral, particularly intravenous orsubcutaneous, delivery according to conventional methods. In general,pharmaceutical formulations will include Cysteine mutant IL-28 or IL-29polypeptide in combination with a pharmaceutically acceptable vehicle,such as saline, buffered saline, 5% dextrose in water, or the like.Formulations may further include one or more excipients, preservatives,solubilizers, buffering agents, albumin to prevent protein loss on vialsurfaces, etc. Methods of formulation are well known in the art and aredisclosed, for example, in Remington: The Science and Practice ofPharmacy, Gennaro, ed., Mack Publishing Co., Easton, Pa., 19^(th) ed.,1995. Cysteine mutant IL-28 or IL-29 will preferably be used in aconcentration of about 10 to 100 μg/ml of total volume, althoughconcentrations in the range of 1 ng/ml to 1000 μg/ml may be used. Fortopical application, such as for the promotion of wound healing, theprotein will be applied in the range of 0.1-10 μg/cm² of wound area,with the exact dose determined by the clinician according to acceptedstandards, taking into account the nature and severity of the conditionto be treated, patient traits, etc. Determination of dose is within thelevel of ordinary skill in the art. Dosing is daily or intermittentlyover the period of treatment. Intravenous administration will be bybolus injection or infusion over a typical period of one to severalhours. Sustained release formulations can also be employed. In general,a therapeutically effective amount of IL-28 or IL-29 Cysteine mutant isan amount sufficient to produce a clinically significant change in thetreated condition, such as a clinically significant change in viral loador immune function, a significant reduction in morbidity, or asignificantly increased histological score.

As an illustration, pharmaceutical formulations may be supplied as a kitcomprising a container that comprises a IL-28 or IL29 polypeptide of thepresent invention. Therapeutic polypeptides can be provided in the formof an injectable solution for single or multiple doses, or as a sterilepowder that will be reconstituted before injection. Alternatively, sucha kit can include a dry-powder disperser, liquid aerosol generator, ornebulizer for administration of a therapeutic polypeptide. Such a kitmay further comprise writ en information on indications and usage of thepharmaceutical composition. Moreover, such information may include astatement that the IL-28 or IL29 polypeptide formulation iscontraindicated in patients with known hypersensitivity to IL-28 or IL29polypeptide.

Within another aspect the present invention provides a method ofproducing an antibody to a polypeptide comprising: inoculating an animalwith a polypeptide selected from the group consisting of SEQ ID NOs:19,21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, and 161, wherein the polypeptide elicits animmune response in the animal to produce the antibody; and isolating theantibody from the animal. Within another aspect the present inventionprovides an antibody (e.g., neutralizing antibody) produced by themethod as disclosed above, wherein the antibody binds to a polypeptideselected from the group consisting of SEQ ID NOs:19, 21, 23, 25, 27, 29,36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, and 161. In one embodiment, the antibody disclosed abovespecifically binds to a polypeptide polypeptide selected from the groupconsisting of SEQ ID NOs:19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161. Withinanother aspect, the present invention provides an antibody or antibodyfragment that specifically binds to a polypeptide as described herein.In one embodiment, the antibody is selected from the group consisting ofa polyclonal antibody, a murine monoclonal antibody a humanized antibodyderived from a murine monoclonal antibody, an antibody fragment, andhuman monoclonal antibody. In one embodiment, the antibody fragment isas described herein, wherein said antibody fragment is selected from thegroup consisting of F(ab′), F(ab), Fab′, Fab, Fv, scFv, and minimalrecognition unit.

Within another aspect, the present invention provides an anti-idiotypeantibody that specifically binds to the antibody as described herein.

As used herein, the term “antibodies” includes polyclonal antibodies,monoclonal antibodies, antigen-binding fragments thereof such as F(ab′)₂and Fab fragments, single chain antibodies, and the like, includinggenetically engineered antibodies. Non-human antibodies may be humanizedby grafting non-human CDRs onto human framework and constant regions, orby incorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. One skilled in the art can generatehumanized antibodies with specific and different constant domains (i.e.,different Ig subclasses) to facilitate or inhibit various immunefunctions associated with particular antibody constant domains.Antibodies are defined to be specifically binding if they bind toCysteine mutant IL-28 or IL-29 polypeptide or protein with an affinityat least 10-fold greater than the binding affinity to control(non-Cysteine mutant IL-28 and IL-29) polypeptide or protein. Theaffinity of a monoclonal antibody can be readily determined by one ofordinary skill in the art (see, for example, Scatchard, Ann. NY Acad.Sci. 51: 660-672, 1949).

Methods for preparing polyclonal and monoclonal antibodies are wellknown in the art (see for example, Hurrell, J. G. R., Ed., MonoclonalHybridoma Antibodies: Techniques and Applications, CRC Press, Inc., BocaRaton, Fla., 1982, which is incorporated herein by reference). Thepolypeptide immunogen may be a full-length molecule or a portionthereof. If the polypeptide portion is “hapten-like”, such portion maybe advantageously joined or linked to a macromolecular carrier (such askeyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanustoxoid) for immunization.

A variety of assays known to those skilled in the art can be utilized todetect antibodies which specifically bind to Cysteine mutant IL-28 orIL-29 polypeptides. Exemplary assays are described in detail in UsingAntibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold SpringHarbor Laboratory Press, 1999. Representative examples of such assaysinclude: concurrent immunoelectrophoresis, radio-immunoassays,radio-immunoprecipitations, enzyme-linked immunosorbent assays (ELISA),dot blot assays, Western blot assays, inhibition or competition assays,and sandwich assays.

For certain applications, including in vitro and in vivo diagnosticuses, it is advantageous to employ labeled antibodies. Suitable directtags or labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent markers, chemiluminescent markers, magneticparticles and the like; indirect tags or labels may feature use ofbiotin-avidin or other complement/anti-complement pairs asintermediates. Antibodies of the present invention may also be directlyor indirectly conjugated to drugs, toxins, radionuclides and the like,and these conjugates used for in vivo diagnostic or therapeuticapplications(e.g., inhibition of cell proliferation). See, in general,Ramakrishnan et al., Cancer Res. 56:1324-1330, 1996.

The present invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Mammalian Expression Plasmids

An expression plasmid containing zcyto20 and zcyto21 was constructed viahomologous recombination. Fragments of zcyto20 and zcyto21 cDNA weregenerated using PCR amplification. The primers for PCR were as follows:

zcyto20/pZMP21: zc40923, and zc43152 SEQ ID NOs:42 and 43, respectively;and zcyto21/pZMP21: zc40922, and zc43153 SEQ ID NOs:2 and 73,respectively.

The PCR reaction mixture was run on a 1% agarose gel and a bandcorresponding to the size of the insert was gel-extracted using aQIAquick™ Gel Extraction Kit (Qiagen, Valencia, Calif.).

The plasmid pZMP21, which was cut with BglII, was used for recombinationwith the PCR insert fragment. Plasmid pZMP21 is a mammalian expressionvector containing an expression cassette having the MPSV promoter, andmultiple restriction sites for insertion of coding sequences; an E. coliorigin of replication; a mammalian selectable marker expression unitcomprising an SV40 promoter, enhancer and origin of replication, a DHFRgene, and the SV40 terminator; and URA3 and CEN-ARS sequences requiredfor selection and replication in S. cerevisiae. It was constructed frompZP9 (deposited at the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209, under Accession No.98668) with the yeast genetic elements taken from pRS316 (deposited atthe American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209, under Accession No. 77145), an internalribosome entry site (IRES) element from poliovirus, and theextracellular domain of CD8 truncated at the C-terminal end of thetransmembrane domain.

One hundred microliters of competent yeast (S. cerevisiae) cells wereindependently combined with 10 μl of the insert DNA and 100 ng of thecut pZMP21 vector above, and the mix was transferred to a 0.2-cmelectroporation cuvette. The yeast/DNA mixture was electropulsed usingpower supply (BioRad Laboratories, Hercules, Calif.) settings of 0.75 kV(5 kV/cm), ∝ohms, and 25 μF. Six hundred μl of 1.2 M sorbitol was addedto the cuvette, and the yeast was plated in a 100-μl and 300 μl aliquotonto two URA-D plates and incubated at 30° C. After about 72 hours, theUra⁺ yeast transformants from a single plate were resuspended in 1 mlH₂O and spun briefly to pellet the yeast cells. The cell pellet wasresuspended in 0.5 ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mMNaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). The five hundred microliters ofthe lysis mixture was added to an Eppendorf tube containing 250 μlacid-washed glass beads and 300 μl phenol-chloroform, was vortexed for 3minutes, and spun for 5 minutes in an Eppendorf centrifuge at maximumspeed. Three hundred microliters of the aqueous phase was transferred toa fresh tube, and the DNA was precipitated with 600 μl ethanol (EtOH)and 30 μl 3M sodium acetate, followed by centrifugation for 30 minutesat maximum speed. The DNA pellet was resuspended in 30 μl TE.

Transformation of electrocompetent E. coli host cells (MC1061) was doneusing 5 μl of the yeast DNA prep and 50 μl of cells. The cells wereelectropulsed at 2.0 kV, 25 PF, and 400 ohms. Following electroporation,1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) was added and then the cells were plated in a 50 μl and 200 μlaliquot on two LB AMP plates (LB broth (Lennox), 1.8% Bacto™ Agar(Difco), 100 mg/L Ampicillin).

The inserts of three clones for each construct were subjected tosequence analysis and one clone for each construct, containing thecorrect sequence, was selected. Larger scale plasmid DNA was isolatedusing a commercially available kit (QIAGEN Plasmid Mega Kit, Qiagen,Valencia, Calif.) according to manufacturer's instructions. The correctconstructs were designated zcyto20/pZMP21 and zcyto21/pZMP21.

Example 2 Expression of Mammalian Constructs in CHO Cells

200 μg of a zcyto20/pZMP21 and zcyto21/pZMP21 construct were digestedwith 200 units of Pvu I at 37° C. for three hours and then wereprecipitated with IPA and spun down in a 1.5 mL microfuge tube. Thesupernatant was decanted off the pellet, and the pellet was washed with1 mL of 70% ethanol and allowed to incubate for 5 minutes at roomtemperature. The tube was spun in a microfuge for 10 minutes at 14,000RPM and the supernatant was aspirated off the pellet. The pellet wasthen resuspended in 750 μl of PF—CHO media in a sterile environment, andallowed to incubate at 60° C. for 30 minutes. CHO cells were spun downand resuspended using the DNA-media solution. The DNA/cell mixture wasplaced in a 0.4 cm gap cuveffe and electroporated using the followingparameters: 950 μF, high capacitance, and 300 V. The contents of thecuvette were then removed and diluted to 25 mLs with PF—CHO media andplaced in a 125 mL shake flask. The flask was placed in an incubator ona shaker at 37° C., 6% CO₂, and shaking at 120 RPM.

Example 3 Purification and Analysis of Zcyto20-CHO Protein

A. Purification of Zcyto20-CHO Protein

Recombinant zcyto20 (IL-28A) protein was produced from a pool ofDXB11-CHO cell lines. Cultures were harvested, and the media weresterile filtered using a 0.2 μm filter.

The purification of zcyto20-CHO protein was achieved by the sequentialuse of a Poros HS 50 column (Applied Biosystems, Framingham, Mass.), aMonolithic WCX column (Isco, Inc., Lincoln, Nebr.), a ToyoPearl Butyl650S column (TosoH, Montgomeryville, Pa.), and a Superdex 75 column(Amersham Biosciences, Piscataway, N.J.). Culture media from DXB111-CHOwere adjusted to pH 6.0 before loading onto a Poros 50 HS column. Thecolumn was washed with 50 mM MES (2-Morpholinoethanesulfonic acid), 100mM NaCl, pH 6 and the bound protein was eluted with a 10 column volumes(CV) linear gradient to 60% of 50 mM MES, 2 M NaCl, pH 6. The elutingfractions were collected and the presence of zcyto20 protein wasconfirmed by SDS-PAGE with a Coomassie staining. This fractionscontaining zcyto20 protein were pooled, diluted with double distilledwater to a conductivity of about 20 mS, and loaded onto a Monolithic WCXcolumn. The column was washed with 93% of 50 mM MES, 100 mM NaCl, pH 6,and 7% of 50 mM MES, 2 M NaCl, pH 6. The bound protein was eluted with a25-CV linear gradient from 7% to 50% of 50 mM MES, 2 M NaCl, pH 6. Theeluting fractions were collected and the presence of zcyto20 protein wasconfirmed by SDS-PAGE with a Coomassie staining. The fractionscontaining zcyto20 protein were pooled, adjusted to 1 M ammonium sulfateand loaded onto a ToyoPearl Butyl 650S column. Zcyto20 was eluted with adecreasing ammonium sulfate gradient and the fractions containing thepure zcyto20 were pooled and concentrated for injection into a Superdex75 column. Fractions containing zcyto20 protein from the gel filtrationcolumn was pooled, concentrated, filtered through a 0.2 μm filter andfrozen at −80° C. The concentration of the final purified protein wasdetermined by a BCA assay (Pierce Chemical Co., Rockford, Ill.) andHPLC-amino acid analysis.

B. SDS-PAGE and Western Blotting Analysis of Zcyto20-CHO Protein

Recombinant zcyto20 protein was analyzed by SDS-PAGE (Nupage 4-12%Bis-Tris, Invitrogen, Carlsbad, Calif.) and Western blot using rabbitanti-zcyto21-CEE-BV IgG as the primary antibody that cross-reacts tozcyto20-CHO protein. The gel was electrophoresed using Invitrogen'sXcell II mini-cell (Carlsbad, Calif.) and transferred to a 0.2 μmnitrocellulose membrane (Bio-Rad Laboratories, Hercules, Calif.) usingInvitrogen's Xcell II blot module according to directions provided inthe instrument manual. The transfer was run at 500 mA for 50 minutes ina buffer containing 25 mM Tris base, 200 mM glycine, and 20% methanol.The membrane was blocked with 10% non-fat dry milk in 1× PBS for 10minutes then probed with the primary antibody in 1× PBS containing 2.5%non-fat dry milk. The blot was labeled for one hour at room temperaturewhile shaking. For the secondary antibody labeling, blot was washedthree times for 10 minutes each with PBS and then probed with goatanti-rabbit IgG-HRP (Pierce Chemical Co., Rockford, Ill.) for one hour.The blot was washed three times with 1× PBS for 10 minutes each anddeveloped using a 1:1 mixture of SuperSignal® ULTRA reagents (PierceChemical Co., Rockford, Ill.) and the signal was captured using aLumi-Imager (Boehringer Mannheim GmbH, Germany).

C. Summary of Protein Purification and Analysis

The purified zcyto20 protein from the CHO media migrated predominantlyas a doublet at approximately 20 kDa and a minor triplet dimer at about38 kDa on a 4-12% Bis-Tris gel under non-reducing conditions. They allcollapsed into a single 20 kDa band under reducing conditions. MSpeptide mapping indicated a mixture of two isomers with respect todisulfide linkage and the presence of O-linked glycosylation site.

Example 4 Purification and Analysis of Zcyto21-CHO Protein

A. Purification of Zcyto21-CHO Protein

Recombinant zcyto21 was produced from stable DXB11-CHO cell lines.Cultures were harvested, and the media were sterile filtered using a 0.2μm filter. Proteins were purified from the conditioned media by startingwith a combination of cationic and anionic exchange chromatographyfollowed by a hydrophobic interaction chromatography and a sizeexclusion chromatography. DXB111-CHO culture media were adjusted to pH6.0 before loading onto a Poros 50 HS column (Applied Biosystems,Framingham, Mass.). The column was washed with 1× PBS, pH 6 and thebound protein was eluted with 5× PBS, pH 8.4. The eluting fraction wascollected and the presence of zcyto21 protein was confirmed by SDS-PAGEwith a Coomassie stain. This fraction was then diluted to a conductivityof 13 mS and its pH adjusted to 8.4 and flowed through a Poros 50 HQcolumn (Applied Biosystems, Framingham, Mass.). The flow-throughcontaining zcyto21 protein were then adjusted to about 127 mS withammonium sulfate and loaded onto a Toyopearl Phenyl 650S column (TosoH,Montgomeryville, Pa.). Zcyto21 protein was eluted with a decreasingammonium sulfate gradient and the fractions containing the pure zcyto21were pooled and concentrated for injection into a Superdex 75 column(Amersham Biosciences, Piscataway, N.J.). The concentration of the finalpurified protein was determined by a BCA assay (Pierce Chemical Co.,Rockford, Ill.) and HPLC-amino acid analysis.

B. SDS-PAGE and Western Blotting Analysis of Zcyto21-CHO Protein

Recombinant zcyto21 protein was analyzed by SDS-PAGE (Nupage 4-12%Bis-Tris, Invitrogen, Carlsbad, Calif.) and Western blot using rabbitanti-zcyto21-CEE-BV IgG as the primary antibody. The gel waselectrophoresed using Invitrogen's Xcell II mini-cell (Carlsbad, Calif.)and transferred to a 0.2 μm nitrocellulose membrane (Bio-RadLaboratories, Hercules, Calif.) using Invitrogen's Xcell II blot moduleaccording to directions provided in the instrument manual. The transferwas run at 500 mA for 50 minutes in a buffer containing 25 mM Tris base,200 mM glycine, and 20% methanol. The transferred blot was blocked with10% non-fat dry milk in 1× PBS for 10 minutes then probed with theprimary antibody in 1× PBS containing 2.5% non-fat dry milk. The blotwas labeled for one hour at room temperature while shaking. For thesecondary antibody labeling, blot was washed three times for 10 minuteseach with PBS and then probed with goat anti-rabbit IgG-HRP (PierceChemical Co., Rockford, Ill.) for one hour. The blot was washed threetimes with 1× PBS for 10 minutes each and developed using a 1:1 mixtureof SuperSignal® ULTRA reagents (Pierce Chemical Co., Rockford, Ill.) andthe signal was captured using a Lumi-Imager (Boehringer Mannheim GmbH,Germany).

C. Summary of Protein Purification and Analysis

The purified zcyto21 protein from the CHO media migrated as two or moreapproximately 28 kDa bands on a 4-12% Bis-Tris gel under both reducingand non-reducing conditions. MS peptide mapping indicated a mixture oftwo isomers with respect to disulfide linkage and the presence of oneN-linked glycosylation and several O-linked glycosylation sites.

Example 5 Identification of IL-29 Forms

Peak fractions from purified pools of IL-29 were digested overnight at37° C. with sequencing grade trypsin (Roche Applied Science,Indianapolis, Ind.) in phosphate buffer at approximately pH 6.3 to limitdisulfide re-arrangement. Each digest was analyzed by reversed-phaseHPLC (Agilent, Palo Alto, Calif.) connected in-line to a quadrupole-timeof flight hybrid mass spectrometer (Micromass, Milford Mass.). Spectrawere collected, converted from mass to charge ratio to mass, andcompared to all theoretical peptides and disulfide-linked peptidecombinations resulting from trypsin digestion of IL-29. Disulfides wereassigned by comparing spectra before and after reduction with assignmentof appropriate masses to disulfide linked peptides in IL-29. Thematerial from fraction #20 showed the disulfide pattern C15-C112 andC49-C145 with C171 observed as a S-glutathionyl cysteine (all referringto SEQ ID NO:4). The material from fraction #51 showed the disulfidepattern C49-C145 and C112-C171 with C15 observed as an S-glutathionylcysteine (referring to SEQ ID NO:4).

Example 6 E. coli Expression Plasmids

Construction of Expression Vector, pTAP237

Plasmid pTAP237 was generated by inserting a PCR-generated linker intothe SmaI site of pTAP186 by homologous recombination. Plasmid pTAP186was derived from the plasmids pRS316 (a Saccharomyces cerevisiae shuttlevector) and pMAL-c2, an E. coli expression plasmid derived from pKK223-3and comprising the tac promoter and the rrnB terminator. Plasmid pTAP186 contains a kanamycin resistance gene in which the Sma I site hasbeen destroyed and has NotI and SfiI sites flanking the yeast ARS-CEN6and URA3 sequences, facilitating their removal from the plasmid bydigestion with NotI. The PCR-generated linker replaced the expressioncoupler sequence in pTAP186 with the synthetic RBS II sequence. It wasprepared from 100 pmoles each of oligonucleotides zc29,740 and zc29,741,as shown in SEQ ID NOS: 44 and 45, respectively, and approximately 5pmoles each of oligonucleotides zc29,736 and zc29,738, as shown in SEQID NOs:46 and 47, respectively. These oligonucleotides were combined byPCR for ten cycles of 94° C. for 30 seconds, 50° C. for 30 seconds, and72° C. for 30 seconds, followed by 4° C. soak. The resulting PCRproducts were concentrated by precipitation with two times the volume of100% ethanol. Pellet was resuspended in 10 μL water to be used forrecombining into the recipient vector pTAP186 digested with SmaI toproduce the construct containing the synthetic RBS II sequence.Approximately 1 μg of the PCR-generated linker and 100 ng of pTAP186digested with SmaI were mixed together and transformed into competentyeast cells (S. cerevisiae). The yeast was then plated onto −URA Dplates and left at room temperature for about 72 hours. Then the Ura+transformants from a single plate were resuspended in 1 mL H₂O and spunbriefly to pellet the yeast cells. The cell pellet was resuspended in0.5 mL of lysis buffer. DNA was recovered and transformed into E. coliMC1061. Clones were screened by colony PCR as disclosed above using 20pmoles each of oligonucleotides zc29,740 and zc29,741, as shown in SEQID NOS: 44 and 45, respectively. Clones displaying the correct size bandon an agarose gel were subject to sequence analysis. The correct plasmidwas designated pTAP237.

Example 7 Codon Optimization of IL-29 Cysteine Mutant

A. Codon Optimization Generation of the IL-29 Wildtype ExpressionConstruct

Native human IL-29 gene sequence was not well expressed in E. colistrain W3110. Examination of the codons used in the IL-29 codingsequence indicated that it contained an excess of the least frequentlyused codons in E. coli with a CAI value equal to 0.206. The CAI is astatistical measure of synonymous codon bias and can be used to predictthe level of protein production (Sharp et al., Nucleic Acids Res.15(3):1281-95, 1987). Genes coding for highly expressed proteins tend tohave high CAI values (>0.6), while proteins encoded by genes with lowCAI values (≦0.2) are generally inefficiently expressed. This suggesteda reason for the poor production of IL-29 in E. coli. Additionally, therare codons are clustered in the second half of the message leading tohigher probability of translational stalling, premature termination oftranslation, and amino acid misincorporation (Kane J F. Curr. Opin.Biotechnol. 6(5):494-500, 1995).

It has been shown that the expression level of proteins whose genescontain rare codons can be dramatically improved when the level ofcertain rare tRNAs is increased within the host (Zdanovsky et al.,Applied Enviromental Microb. 66:3166-3173, 2000; You et al,.Biotechniques 27:950-954, 1999). The pRARE plasmid carries genesencoding the tRNAs for several codons that are rarely used E. coli(argu, argw, leuW, proL, ileX and glyT). The genes are under the controlof their native promoters (Novy, ibid.). Co-expression with pRAREenhanced IL-29 production in E. coli and yield approximately 200 mg/L.These data suggest that re-resynthesizing the gene coding for IL-29 withmore appropriate codon usage provides an improved vector for expressionof large amounts of IL-29.

The codon optimized IL-29 coding sequence was constructed from sixteenoverlaping oligonucleotides: zc44,566 (SEQ ID NO:48), zc44,565 (SEQ IDNO:49), zc44,564 (SEQ ID NO:50), zc44,563 (SEQ ID NO:51), zc44,562 (SEQID NO:52), zc44,561 (SEQ ID NO:53), zc44,560 (SEQ ID NO:54), zc244,559(SEQ ID NO:55), zc44,558 (SEQ ID NO:56), zc44,557 (SEQ ID NO:57). Primerextension of these overlapping oligonucleotides followed by PCRamplication produced a full length IL-29 gene with codons optimized forexpression in E. col. The final PCR product was inserted into expressionvector pTAP237 by yeast homologous recombination. The expressionconstruct was extracted from yeast and transformed into competent E.coli MC1061. Clones resistance to kanamycin were identified by colonyPCR. A positive clone was verified by sequencing and subsequentlytransformed into production host strain W3110. The expression vectorwith the optimized IL-29 sequence was named pSDH184. The resulting genewas expressed very well in E. coli. expression levels with the newconstruct increased to around 250 mg/L.

B. Generation of the Codon Optimized Zcyto21 C172S Cysteine MutantExpression Construct

The strategy used to generate the zcyto21 C172S Cysteine mutant is basedon the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Primerswere designed to introduce the C172S mutation based on manufacturer'ssuggestions. These primers were designated ZG44,340 (SEQ ID NO:58) andZG44,341 (SEQ ID NO:59). PCR was performed to generate the zcyto21 C172SCysteine mutant according to QuikChange Mutagenesis instructions. Fiveidentical 50 μl reactions were set-up. 2.5 μl pSDH175 (missing yeastvector backbone sequence) DNA was used as template per reaction. A PCRcocktail was made up using the following amounts of reagents: 30 μl 10×PCR buffer, 125 ng (27.42 μl) ZG44,340, 125 ng (9.18 μl) ZG44,341, 6 μldNTP, 6 μl Pfu Turbo polymerase (Stratagene, La Jolla, Calif.), and206.4 μl water. 47.5 μl of the cocktail was aliquotted into eachreaction. The PCR conditions were as follows: 1 cycle of 95° C. for 30seconds followed by 16 cycles of 95° C. for 30 seconds, 55° C. for 1minute, 68° C. for 7 minutes, followed by 1 cycle at 68° C. for 7minutes, and ending with a 4° C. hold. All five PCR reactions wereconsolidated into one tube. As per manufacturer's instructions, 5 μlDpnI restriction enzyme was added to the PCR reaction and incubated at37° C. for 2 hours. DNA was precipitated my adding 10% 3 Molar SodiumAcetate and two volumes of 100% ethanol. Precipitation was carried-outat −20° C. for 20 minutes. DNA was spun at 14,000 rpm for 5 minutes andpellet was speed-vac dried. DNA pellet was resuspended in 20 μl water.DNA resulting from PCR was transformed into E.coli strain DH10B. 5 μlDNA was mixed with 40 μl ElectroMAX DH10B cells (Invitrogen). Cells andDNA mixture were then electroporated in a 0.1 cm cuvette (Bio-Rad) usinga Bio-Rad Gene Pulser II™ set to 1.75 kV, 100Ω, and 25 μF.Electroporated cells were then outgrown at 37° C. for 1 hour. Mixturewas plated on an LB+25 μg/ml kanamycin plate and incubated at 37° C.overnight. Ten clones were screened for presence of zcyto21 C172Sinsert. DNA was isolated from all ten clones using the QIAprep™ SpinMiniprep Kit (Qiagen, Valencia, Calif.) and analyzed for presence ofinsert by cutting with XbaI and PstI restriction enzymes. Nine clonescontained insert and were sequenced to insure the zcyto21 C172S mutationhad been introduced. A clone was sequence verified and was subsequentlylabeled pSDH188.

Example 8 E. coli IL-29 Expression Construct

A DNA fragment of IL-29 containing the wildtype sequence was isolatedusing PCR. Primers zc41,212 (SEQ ID NO: 60) containing 41 base pair (bp)of vector flanking sequence and 24 bp corresponding to the aminoterminus of IL-29, and primer zc41,041 (SEQ ID NO:61) contained 38 bpcorresponding to the 3′ end of the vector which contained the zcyto21insert were used in the reaction. The PCR conditions were as follows: 25cycles of 94° C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for 1minute; followed by a 4° C. soak. A small sample (2-4 μL) of the PCRsample was run on a 1% agarose gel with 1× TBE buffer for analysis, andthe expected band of approximately 500 bp fragment was seen. Theremaining volume of the 100 μL reaction was precipitated with 200 μLabsolute ethanol. The pellet was resuspended in 10 μL water to be usedfor recombining into recipient vector pTAP238 cut with SmaI to producethe construct encoding the zcyto21 as disclosed above. The clone withcorrect sequence was designated as pTAP377. Clone pTAP377 was digestedwith Not1/Nco1 (10 μl DNA, 5 μl buffer 3 New England BioLabs, 2 μL Not1, 2 μL Nco1, 31 μL water for 1 hour at 37° C.) and religated with T4DNA ligase buffer (7 μL of the previous digest, 2 μL of 5× buffer, 1 μLof T4 DNA ligase). This step removed the yeast sequence, CEN-ARS, tostreamline the vector. The pTAP337 DNA was diagnostically digested withPvu2 and Pst1 to confirm the absence of the yeast sequence. P/taP377 DNAwas transformed into E. coli strain W3110/pRARE, host strain carryingextra copies of rare E. coli tRNA genes.

Example 9 E. coli IL-28A Expression Construct

A DNA fragment containing the wildtype sequence of zcyto20 (as shown inSEQ ID NO: 1) was isolated using PCR. Primers zc43,431 (SEQ ID NO:62)containing 41 bp of vector flanking sequence and 24 bp corresponding tothe amino terminus of zcyto20, and primer zc43,437 (SEQ ID NO:63)contained 38 bp corresponding to the 3′ end of the vector whichcontained the zcyto20 insert. The PCR conditions were as follows: 25cycles of 94° C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for 1minute; followed by a 4° C. soak. A small sample (2-4 μL) of the PCRsample was run on a 1% agarose gel with 1× TBE buffer for analysis, andthe expected band of approximately 500 bp fragment was seen. Theremaining volume of the 100 μL reaction was precipitated with 200 μLabsolute ethanol. The pellet was resuspended in 10 μL water to be usedfor recombining into recipient vector pTAP238 cut with SmaI to producethe construct encoding the zcyto20 as disclosed above. The clone withcorrect sequence was designated as pYEL7. It was digested with Not1/Nco1(10 μl DNA, 5 μl buffer 3 New England BioLabs, 2 μL Not1, 2 μL Nco1, 31μL water for 1 hour at 37° C.) and religated with T4 DNA ligase buffer(7 μL of the previous digest, 2 μL of 5× buffer, 1 μL of T4 DNA ligase).This step removed the yeast sequence, CEN-ARS, to streamline the vector.The relegated pYEL7 DNA was diagnostically digested with Pvu2 and Pst1to confirm the absence of the yeast sequence. PYEL7 DNA was transformedinto E. coli strain W3110/pRARE.

Example 10 Zcyto21 C172S Cysteine Mutant Expression Construct

The strategy used to generate the zcyto21 C172S Cysteine mutant (SEQ IDNO: 28) is based on the QuikChange® Site-Directed Mutagenesis Kit(Stratagene, La Jolla, Calif.). Primers were designed to introduce theC172S mutation based on manufacturer's suggestions. These primers weredesignated ZG44,327 and ZG44,328 (SEQ ID NOs:64 and 65, respectively).PCR was performed to generate the zcyto21 C172S Cysteine mutantaccording to QuikChange Mutagenesis instructions. Five identical 50 μlreactions were set-up. 2.5 μl pTAP377 (missing yeast vector backbonesequence) DNA was used as template per reaction. A PCR cocktail was madeup using the following amounts of reagents: 30 μl 10× PCR buffer, 125 ng(27.42 μl) ZG44,327 (SEQ ID NO: 64), 125 ng (9.18 μl) ZG44,328 (SEQ IDNO: 65), 6 μl dNTP, 6 μl Pfu Turbo polymerase (Strategene), and 206.4 μlwater. 47.5 μl of the cocktail was aliquotted into each reaction. ThePCR conditions were as follows: 1 cycle of 95° C. for 30 secondsfollowed by 16 cycles of 95° C. for 30 seconds, 55° C. for 1 minute, 68°C. for 7 minutes, followed by 1 cycle at 68° C. for 7 minutes, andending with a 4° C. hold. All five PCR reactions were consolidated intoone tube. As per manufacturer's instructions, 5 μl DpnI restrictionenzyme was added to the PCR reaction and incubated at 37° C. for 2hours. DNA was precipitated my adding 10% 3 Molar Sodium Acetate and twovolumes of 100% ethanol (Aaper Alcohol, Shelbyville, Ky.). Precipitationwas carried-out at −20° C. for 20 minutes. DNA was spun at 14,000 rpmfor 5 minutes and pellet was speed-vac dried. DNA pellet was resuspendedin 20 μl water. DNA resulting from PCR was transformed into E. colistrain DH10B. 5 μl DNA was mixed with 40 μl ElectroMAX DH10B cells(Invitrogen, Carlsbad, Calif.). Cells and DNA mixture were thenelectroporated in a 0.1 cm cuvette (Bio-Rad, Hercules, Calif.) using aBio-Rad Gene Pulser II™ set to 1.75 kV, 100Ω, and 25 μF. Electroporatedcells were then outgrown at 37° C. for 1 hour. Mixture was plated on anLB+25 μg/ml kanamycin plate and incubated at 37° C. overnight. Tenclones were screened for presence of IL-29 insert. DNA was isolated fromall ten clones using the QIAprep™ Spin Miniprep Kit (Qiagen) andanalyzed for presence of insert by cutting with XbaI (Roche) and PstI(New England Biolabs) restriction enzymes. Nine clones contained insertand were sequenced to insure the zcyto21 C172S mutation had beenintroduced. A clone (isolet #6) was sequence verified and wassubsequently labeled pSDH171. A similar strategy can be implemented togenerate a zcyto21 C15S mutant.

Example 11 Zcyto20 C49S Cysteine Mutant Expression Construct

The zcyto20 C49S Cysteine mutant coding sequence was generated byoverlap PCR (SEQ ID NO:20). The first 187 bases of the wildtype IL-28Asequence (SEQ ID NO:1) was generated by PCR amplification using pYEL7(SEQ ID NO:67) as template and oligonucleotide primers zc43,431 (SEQ IDNO: 62) and zc45,399 (SEQ ID NO:66). The second DNA fragment from base105 to 531 was generated by PCR amplification using pYEL7 (SEQ ID NO:67)as template and oligonucleotide primers zc45,398 (SEQ ID NO: 68) andzc43,437 (SEQ ID NO:63). Primers zc45,399 (SEQ ID NO:66) and zc45,398(SEQ ID NO:68) contained the specific modified sequence which changedthe cysteine 49 to a serine. These two PCR products were combined andPCR overlap amplified using oligonucleotide primers zc43,431 (SEQ IDNO:62) and zc43,437 (SEQ ID NO:63). The final PCR product was insertedinto expression vector pTAP238 by yeast homologous recombination(Raymond et al. Biotechniques. January 26(1):134-8, 140-1, 1999). Theexpression construct was extracted from yeast and transformed intocompetent E. coli DH10B. Kanamycin resistant clones were screened bycolony PCR. A positive clone was verified by sequencing and subsequentlytransformed into production host strain W3110/pRARE. The expressionconstruct with the zcyto20 C49S Cysteine mutant coding sequence wasnamed pCHAN9.

Example 12 Zcyto20 C51S Cysteine Mutant Expression Construct

The zcyto20 C51S Cysteine mutant coding sequence was generated byoverlap PCR (SEQ ID NO:24). The first 193 bases of the wildtype IL-28Asequence was generated by PCR amplification using pYEL7 (SEQ ID NO:67)as template and oligonucleotide primers zc43,431 (SEQ ID NO:62) andzc45,397 (SEQ ID NO:63). The second DNA fragment from base 111 to 531was generated by PCR amplification using pYEL7 (SEQ ID NO:67) astemplate and oligonucleotide primers zc45,396 (SEQ ID NO:70) andzc43,437 (SEQ ID NO:63). Primers zc45,397 (SEQ ID NO:69) and zc45,396(SEQ ID NO:70) contained the specific modified sequence which changedthe cysteine51 to a serine. These two PCR products were combined and PCRoverlap amplified using oligonucleotide primers zc43,431 (SEQ ID NO:62)and zc43,437 (SEQ ID NO:63). The final PCR product was inserted into ourin-house expression vector pTAP238 by yeast homologous recombination(Raymond et al. supra). The expression construct was extracted fromyeast and transformed into competent E. coli DH10B. Kanamycin resistantclones were screened by colony PCR. A positive clone was verified bysequencing and subsequently transformed into production host strainW3110/pRARE. The expression construct with the zcyto20 C50S Cysteinemutant coding sequence was named pCHAN10.

Example 13 Expression of I1-28A, IL-29 and Cys to Ser Cysteine Mutantsin E. coli

In separate experiments, E. coli transformed with each of the expressionvectors described in Examples 6-9 were inoculated into 100 mL SuperbrothII medium (Becton Dickinson, San Diego, Calif.) with 0.01% Antifoam 289(Sigma Aldrich, St. Louis, Mo.), 30 μg/ml kanamycin, 35 μg/mlchloramphenicol and cultured overnight at 37° C. A 5 mL inoculum wasadded to 500 mL of same medium in a 2 L culture flask which was shakenat 250 rpm at 37° C. until the culture attained an OD600 of 4. IPTG wasthen added to a final concentration of 1 mM and shaking was continuedfor another 2.5 hours. The cells were centrifuged at 4,000×g for 10 minat 4° C. The cell pellets were frozen at −80° C. until use at a latertime.

Example 14 Refolding and Purification of IL-28

A. Inclusion Body Preparation

Human wildtype IL-29 was expressed in E. coli strain W3110 as inclusionbodies as described above. A cell pellet from a fed-batch fermentationwas resuspended in 50 mM Tris, pH 7.3. The suspension was passed throughan APV-Gaulin homogenizer (Invensys APV, Tonawanda, N.Y.) three times at8000 psi. The insoluble material was recovered by centrifugation at15,000 g for 30 minutes. The pellet was washed consecutively with 50 mMTris, 1% (v/v) Triton X100, pH 7.3 and 4 M Urea. The inclusion body wasthen dispersed in 50 mM Tris, 6 M guanidine hydrochloride, 5 mM DTT atroom temperature for 1 hour. The material was then centrifuged at 15,000g for 1 hour. The supernatant from this step contains reduced solubleIL-29.

B. Refolding

The solubilized IL-29 was diluted slowly into 50 mM Tris, pH 8, 0.75 MArginine, 0.05% PEG3350, 2 mM MgCl₂, 2 mM CaCl₂, 0.4 mM KCl, 10 mM NaCl,4 mM reduced Glutathione, 0.8 mM oxidized Glutathione at roomtemperature while stirring. The final concentration of IL-29 in therefolding buffer was 0.1 mg/ml. The refolding mixture was left at roomtemperature overnight. Concentrated acetic acid was then used to adjustthe pH of the suspension to 5. The suspension was then filtered througha 0.2 μm filter. RP-HPLC analysis of the refolding mixture showed twoprominent peaks.

C. Purification

The refolding mixture was in-line diluted (1:2) with 50 mM NaOAc at pH 5and loaded onto a Pharmacia SP Sepharose Fast Flow cation exchangecolumn (North Peapack, N.J.). The column was washed with 3 columnvolumes of 50 mM NaOAc, 400 mM NaCl, pH 5. The bound IL-29 was elutedwith 50 mM NaOAc, 1.4 M NaCl, pH 5. Solid (H₄)₂SO₄ was added to theelute pool of the cation exchange step so that the final concentrationof (H₄)₂SO₄ was 0.5 M. The material was then loaded onto a ToyoPearlPhenyl 650S HIC column (Tosoh Biosep, Montgomery, Pa.). The column wasthen washed with 3 column volumes of 50 mM NaOAc, 1 M (H₄)₂SO₄, pH 5. Alinear gradient of 10 column volumes from 50 mM NaOAc, 1 M (H₄)₂SO₄, pH5 to 50 mM NaOAc, pH 5 was used to elute the bound zcyto21. Fractionswere collected of the elute. Two prominent peaks were observed in thisstep. RP-HPLC analysis of the elute fractions was performed. Twoproducts corresponding to two disulfide bond isomers were produced afterfinal buffer exchange into PBS, pH 7.3.

Example 15 Refolding and Purification of IL-29 Cysteine Mutant

As described in Example 3, purification of IL-29 produced two disulfidebond isomers. A HIC FPLC step was employed to separate the two forms.The separation was not baseline resolved. Severe “Peak Shaving” had tobe used to obtain substantially pure isomers (>95%). The yield for thisstep and by extension for the whole process suffered. The final yieldswere 8% and 9% for the C15-C112 form and C112-C171 form respectively.Wildtype IL-29 produced in CHO and baculovirus (BV) systems also showedsimilar phenomena. It was established that the C15-C112 form of theisomer is homologous in disulfide bond patterns to type I INF's. TheC15-C112 form also demonstrated 30-fold higher bioactivity than theC112-C171 form in an ISRE assay (see below).

Refolding and Purification of Zcyto21 Cys172Ser Mutein

The inclusion body preparation, refolding and purification of zcyto21C172S polypeptide (SEQ ID NO:29) is essentially the same as those ofIL-29 wild-type (SEQ ID NO:4). RP-HPLC analysis of the refolding mixtureof the mutein showed only one prominent peak corresponding to theC15-C112 form of the wild-type IL-29. Subsequent HIC chromatography showonly a single peak. It was therefore unnecessary to employ severe “peakshaving”. The final yield for the entire process is close to 50%. Thezcyto21 Cys172Ser polypeptide (SEQ ID NO:29) showed equivalentbioactivity to the C15-C112 form of wild-type IL-29 in ISRE assay shownin Example 16.

Example 16 Antiviral Activity: Cytopathic Effect in Hela and L929 Cells

Initial functional assays for antiviral activity were conducted usingconditioned media from transiently transfected human embryonal kidney(HEK) cells. Production of this conditioned medium is described asfollows. A full-length cDNA for human or murine IL-28A, IL-28B, or IL-29was cloned into the pzp7Z vector using standard procedures. The human ormurine IL-28A, IL-28B, or IL-29 constructs were transfected into 293 HEKcells. Briefly, for each construct 700,000 cells/well (6 well plates)were plated approximately 18 h prior to transfection in 2 millilitersDMEM+10% fetal bovine serum. Per well, 1.5 micrograms human or murineIL-28A, IL-28B, or IL-29 DNA and 0.5 micrograms pIRES2-EGFP DNA(Clontech) were added to 6 microliters Fugene 6 reagent (RocheBiochemicals) in a total of 100 microliters DMEM. Two microgramspIRES2-EGFP DNA alone was used as a negative control. These transfectionmixtures were added 30 minutes later to the pre-plated 293 cells.Twenty-four hours later the cell media were removed and DMEM+0.1% bovineserum albumin was added. Conditioned media was collected after 48 hours,filtered through a 0.45 micron filter and used for antiviral andreporter assays.

Antiviral Assays were carried out using human cervical carcinoma cells(HeLa) and mouse fibroblast cells (L929). On the first day, conditionedmedium containing human or murine IL-28A, IL-28B, or IL-29 was dilutedand plated with 50,000 cells in a 96-well flat bottom microtiter plate.Following a 24-hour incubation at 37° C., the medium was removed andreplaced with medium containing encephelomyocarditis virus at amultiplicity of infection of 0.1. The cells were again incubated for 24hours at 37° C. Culture wells were then scored visually on a 4-pointscale for the presence of cytopathic effect, which was then converted to% CPE as shown in Table 7. Conditioned medium from cells transfectedwith GFP alone and purified human interferon-a-2a or murineinterferon-alpha were included as controls. TABLE 7 Determination ofCytopathic Effect Designation Observation of Cytopathic Effect (CPE) −No CPE +/− Possible CPE (about 1% of monolayer surface) + CPE limited toone plaque (about 5% of the surface) +1  CPE is limited to threeplaques, affecting less than 25% of the monolayer 1 25% CPE 1-2 37% CPE2 50% CPE 2-3 62% CPE 3 75% CPE 3-4 87% CPE 4 100% CPE

Table 8 shows that conditioned medium containing human or murine IL-28A,IL-28B, or IL-29 inhibited viral infection (% CPE) in HeLa cells in adose-dependent manner, while control GFP conditioned medium failed tosignificantly block the appearance of cytopathic effect. As shown inTable 9, conditioned medium containing human or murine IL-28A, IL-28B,or IL-29 did not inhibit viral infection in L929 cells. In bothexperiments purified interferon showed positive antiviral activity.TABLE 8 Percentage Cytopathic Effect of human or murine IL-28A, IL-28B,or IL-29 in HeLa Cells using Conditioned Medium (CM) zcyto24 zcyto25zcyto20 zcyto21 zcyto22 mouse mouse Relative CM Control IL-28A IL-29IL-28B IL-28 IL-28 hIFN- hIFN-a-2a Concentration GFP (CM) (CM) (CM) (CM)(CM) a-2a Concentration No Add 87 87 87 87 87 87 87 0 ng/ml .008X 87 1056 0 0 10 15 .0001 ng/ml    .0156X 87 2.5 31 0 0 5 8.3 .001 ng/ml  .0325X 87 5 10 0 0 5 1.7 .01 ng/ml   .0625X 87 2.5 10 0 0 0 0 .1 ng/ml .125X 87 0 5 0 0 0 0 1 ng/ml .25X 87 0 0 0 0 0 0 10 ng/ml  .5X 87 0 0 00 0 0 100 ng/ml 

TABLE 9 Percentage Cytopathic Effect of human or murine IL-28A, IL-28B,or IL-29 in L929 Cells using Conditioned Medium (CM) Relative CM Controlzcyto20 zcyto21 zcyto22 zcyto24 zcyto25 mIFN- mIFN-alpha Conc. GFP (CM)(CM) (CM) (CM) (CM) alpha Conc. No Add 87 87 87 87 87 87 87 0 ng/ml.008X 87 87 87 87 87 87 87 .0001 ng/ml    .0156X 87 87 87 87 87 87 87.001 ng/ml   .0325X 87 87 87 87 87 87 87 .01 ng/ml   .0625X 87 87 87 8787 87 58 .1 ng/ml  .125X 87 87 87 87 87 87 6.7 1 ng/ml .25X 87 87 87 8787 87 0 10 ng/ml  .5X 87 87 87 87 87 87 0 100 ng/ml 

Example 17 Signaling Via Interferon-Response Pathway

Interaction of type 1 interferons with their specific receptor leads toinduction of a number of genes responsible for theirantiviral/antiproliferative activity. These include 2′-5′ oligoadenylatesynthetase (2-5 OAS), double-stranded RNA dependent Pkr kinase (Pkr),phospholipid scramblase, and intercellular adhesion molecule-1 (ICAM-1).Induction of genes with as yet unknown function, such as a 56 kDainterferon stimulated gene product (ISG-56 k), also occurs. To determineif some or all of these genes are induced upon treatment of cells withIL-28A, human Daudi B lymphoid cells were treated for 72 hours withconditioned medium from Sf9 cells infected with baculovirus expressingIL-28A. Conditioned medium from Sf9 cells infected with wild-typebaculovirus was used as a negative control. Following treatment cellswere collected and lysed for isolation of total RNA. One microgram oftotal RNA was converted to cDNA using reverse transcriptase and used asa template for polymerase chain reaction using oligonucleotide primersspecific for the human interferon-stimulated genes described above.Oligonucleotide primers for human glycerol-3-phosphate dehydrogenase(G3PDH) were used as a non-interferon stimulated gene control. Theresults show clear induction of ISG-56 k, Pkr, 2-5 OAS and phospholipidscramblase following treatment of cells with IL-28A. No induction wasseen for ICAM-1 or the non-interferon stimulated gene control, G3PDH.

Example 18 Signal Transduction Reporter Assay

A signal transduction reporter assay can be used to determine thefunctional interaction of human and mouse IL-28 and IL-29 with the IL-28receptor. Human embryonal kidney (HEK) cells are transfected with areporter plasmid containing an interferon-stimulated response element(ISRE) driving transcription of a luciferase reporter gene in thepresence or absence of pZP7 expression vectors containing cDNAs forclass II cytokine receptors (including human DIRS1, IFNαR1, IFNαR2 andIL-28 receptor). Luciferase activity following stimulation oftransfected cells with class II ligands (including IL-28A (SEQ ID NO:2),IL-29 (SEQ ID NO:4), IL-28B (SEQ ID NO:6), zcyto10, huIL10 andhuIFNa-2a) reflects the interaction of the ligand with transfected andnative cytokine receptors on the cell surface. The results and methodsare described below.

Cell Transfections

293 HEK cells were transfected as follows: 700,000 293 cells/well (6well plates) were plated approximately 18 h prior to transfection in 2milliliters DMEM+10% fetal bovine serum. Per well, 1 microgrampISRE-Luciferase DNA (Stratagene), 1 microgram cytokine receptor DNA and1 microgram pIRES2-EGFP DNA (Clontech,) were added to 9 microlitersFugene 6 reagent (Roche Biochemicals) in a total of 100 microlitersDMEM. Two micrograms pIRES2-EGFP DNA was used when cytokine receptor DNAwas not included. This transfection mix was added 30 minutes later tothe pre-plated 293 cells. Twenty-four hours later the transfected cellswere removed from the plate using trypsin-EDTA and replated atapproximately 25,000 cells/well in 96 well microtiter plates.Approximately 18 h prior to ligand stimulation, media was changed toDMEM+0.5% FBS.

Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37° C. in DMEM+0.5% FBS, transfected cells werestimulated with dilutions (in DMEM+0.5% FBS) of the following class IIligands; IL-28A, IL-29, IL-28B, zcyto10, huIL10 and huIFNa-2a. Followinga 4-hour incubation at 37° C., the cells were lysed, and the relativelight units (RLU) were measured on a luminometer after addition of aluciferase substrate. The results obtained are shown as the foldinduction of the RLU of the experimental samples over the medium alonecontrol (RLU of experimental samples/RLU of medium alone=foldinduction). Table 10 shows that IL-28A, IL-29, and IL-28B induce ISREsignaling in 293 cells transfected with ISRE-luciferase giving a 15 to17-fold induction in luciferase activity over medium alone. The additionof IL-28 receptor alpha subunit DNA (SEQ ID NO:11), using the endogenousCRF2-4 (SEQ ID NO:71) to the transfection mix results in a 6 to 8-foldfurther induction in ISRE signaling by IL-28A, IL-29, and IL-28B givinga 104 to 125-fold total induction. None of the other transfected classII cytokine receptor DNAs resulted in increased ISRE signaling. Theseresults indicate that IL-28A, IL-29, and IL-28B functionally interactwith the IL-28 cytokine receptor. Table 10 also shows that huIFNa-2a caninduce ISRE signaling in ISRE-luciferase transfected 293 cells giving a205-fold induction of luciferase activity compared to medium alone.However, the addition of IL-28 receptor DNA to the transfection leads toan 11-fold reduction in ISRE-signaling (compared to ISRE-luciferase DNAalone), suggesting that IL-28 receptor over-expression negativelyeffects interferon signaling, in contrast to the positive effects ofIL-28 receptor over-expression on IL-28A, IL-29, and IL-28B signaling.TABLE 10 Interferon Stimulated Response Element (ISRE) Signaling ofTransfected 293 Cells Following Class II Cytokine Stimulation (FoldInduction) Ligand ISRE-Luc. ISRE-Luc./IL-28R IL-28A (125 ng/ml) 15 125IL-29 (125 ng/ml) 17 108 IL-28B (125 ng/ml) 17 104 HuIFNa-2a (100 ng/ml)205 18 Zcyto10 (125 ng/ml) 1.3 1 HuIL10 (100 ng/ml) 1 0.5

Example 19 Signal Transduction Assays with IL-29 Cysteine Mutants

Cell Transfections

To produce 293 HEK cells stably overexpressing human IL-28 receptor, 293cells were transfected as follows: 300,000 293 cells/well (6 wellplates) were plated approximately 6 h prior to transfection in 2milliliters DMEM+10% fetal bovine serum. Per well, 2 micrograms of apZP7 expression vector containing the cDNA of human IL-28 receptor alphasubunit (SEQ ID NO: 11) was added to 6 microliters Fugene 6 reagent(Roche Biochemicals) in a total of 100 microliters DMEM. Thistransfection mix was added 30 minutes later to the pre-plated 293 cells.Forty-eight hours later the transfected cells were placed under 2microgram/milliliter puromicin selection. Puromicin resistant cells werecarried as a population of cells.

The 293 HEK cells overexpressing human IL-28 receptor were transfectedas follows: 700,000 293 cells/well (6 well plates) were platedapproximately 18 h prior to transfection in 2 milliliters DMEM+10% fetalbovine serum. Per well, 1 microgram KZ 157 containing aninterferon-stimulated response element (ISRE) driving transcription of aluciferase reporter gene were added to 3 microliters Fugene 6 reagent(Roche Biochemicals) in a total of 100 microliters DMEM. Thistransfection mix was added 30 minutes later to the pre-plated 293HEKcells. Forty-eight hours later the transfected cells were removed fromthe plate using trypsin-EDTA and replated in 500 micrograms/ml G418(Geneticin, Life Technologies). Puromycin and G418 resistant cells werecarried as a population of cells.

Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: 293HEKcells overexpressing human IL-28 receptor and containing KZ157 weretreated with trypsin-EDTA and replated at approximately 25,000cells/well in 96 well microtiter plates. Approximately 18 h prior toligand stimulation, media was changed to DMEM+0.5% FBS.

Following an 18 h incubation at 37° C. in DMEM+0.5% FBS, transfectedcells were stimulated with dilutions (in DMEM+0.5% FBS) of the differentforms of E.coli-derived zcyto21 containing different cysteine bindingpatterns. Following a 4-hour incubation at 37° C., the cells were lysed,and the relative light units (RLU) were measured on a luminometer afteraddition of a luciferase substrate. The results obtained are shown asthe fold induction of the RLU of the experimental samples over themedium alone control (RLU of experimental samples/RLU of mediumalone=fold induction).

Table 11 shows that C1-C3 form (C16-C113) of wild-type E. coli-derivedIL-29 is better able to induce ISRE signaling than wild-type C3-C5 form(C113-C172) or a mixture of wild-type C1-C3 form and C3-C5 form(C16-C113, C113-C172), all referring to SEQ ID NO:15.

Table 12 shows that C1-C3 (C16-C113) of wild-type E. coli-derived IL-29and C1-C3 (C16-C113; SEQ ID NO:15) of Cysteine mutant (C172S) E.coli-derived IL-29 (SEQ ID NO:29) are equally able to induce ISREsignaling in 293HEK cells overexpressing human IL-28 receptor. TABLE 11ISRE Signaling by different forms of E. coli-derived IL-29 (FoldInduction) Cytokine Concentration C1-C3 form C3-C5 form Mixture of C1-C3(ng/ml) (C16-C113) (C113-C172) and C3-C5 100 36 29 34 10 38 25 35 1 3212 24 0.1 10 2 5 0.01 3 1 1 0.001 1 1 1

TABLE 12 ISRE Signaling by different forms of E. coli-derived IL-29(Fold Induction) Cytokine Cysteine mutant Concentration Wild-type C172S(ng/ml) C1-C3 C1-C3 1000 9.9 8.9 100 9.3 8.7 10 9.3 8.1 1 7.8 7 0.1 4.63.3 0.01 1.9 1.5 0.001 1.3 0.9

Example 20 Induction of IL-28A, IL-29 IL-28B by Poly I:C and ViralInfection

Freshly isolated human peripheral blood mononuclear cells were grown inthe presence of polyinosinic acid-polycytidylic acid (poly I:C; 100μg/ml) (SIGMA; St. Louis, Mo.), encephalomyocarditis virus (EMCV) withan MOI of 0.1, or in medium alone. After a 15 h incubation, total RNAwas isolated from cells and treated with RNase-free DNase. 100 ng totalRNA was used as template for one-step RT-PCR using the SuperscriptOne-Step RT-PCR with Platinum Taq kit and gene-specific primers assuggested by the manufacturer (Invitrogen).

Low to undetectable amounts of human IL-28A, IL-28B, and IL-29, IFN-αand IFN-β RNA were seen in untreated cells. In contrast, the amount ofIL-28A, IL-29, IL-28B RNA was increased by both poly I:C treatment andviral infection, as was also seen for the type I interferons. Theseexperiments indicate that IL-28A, IL-29, IL-28B, like type Iinterferons, can be induced by double-stranded RNA or viral infection.

Example 21 IL-28, IL-29 Signaling Activity Compared to IFNα in HepG2Cells

A. Cell Transfections

HepG2 cells were transfected as follows: 700,000 HepG2 cells/well (6well plates) were plated approximately 18 h prior to transfection in 2milliliters DMEM+10% fetal bovine serum. Per well, 1 microgrampISRE-Luciferase DNA (Stratagene) and 1 microgram pIRES2-EGFP DNA(Clontech,) were added to 6 microliters Fugene 6 reagent (RocheBiochemicals) in a total of 100 microliters DMEM. This transfection mixwas added 30 minutes later to the pre-plated HepG2 cells. Twenty-fourhours later the transfected cells were removed from the plate usingtrypsin-EDTA and replated at approximately 25,000 cells/well in 96 wellmicrotiter plates. Approximately 18 h prior to ligand stimulation, mediawas changed to DMEM+0.5% FBS.

B. Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37° C. in DMEM+0.5% FBS, transfected cells werestimulated with 100 ng/ml IL-28A, IL-29, IL-28B, zcyto24, zcyto25 andhuIFN-α2a ligands. Following a 4-hour incubation at 37° C. degrees, thecells were lysed, and the relative light units (RLU) were measured on aluminometer after addition of a luciferase substrate. The resultsobtained are shown as the fold induction of the RLU of the experimentalsamples over the medium alone control (RLU of experimental samples/RLUof medium alone=fold induction). Table 13 shows that IL-28A, IL-29,IL-28B, zcyto24 and zcyto25 induce ISRE signaling in human HepG2 livercells transfected with ISRE-luciferase. TABLE 13 Fold Induction ofCytokine-dependent ISRE Signaling in HepG2 Cells Cytokine Fold InductionIL-28A 5.6 IL-29 4 IL-28B 5.8 Zcyto24 4.7 Zcyto25 3 HuIFN-a2a 5.8

Example 22 IL-29 Antiviral Activity Compared to IFNα in HepG2 Cells

An antiviral assay was adapted for EMCV (American Type CultureCollection # VR-129B, Manassas, Va.) with human cells (Familletti, P.,et al., Methods Enzym. 78: 387-394, 1981). Cells were plated withcytokines and incubated 24 hours prior to challenge by EMCV at amultiplicity of infection of 0.1 to 1. The cells were analyzed forviability with a dye-uptake bioassay 24 hours after infection (Berg, K.,et al., Apmis 98: 156-162, 1990 ). Target cells were given MTT andincubated at 37° C. for 2 hours. A solubiliser solution was added,incubated overnight at 37° C. and the optical density at 570 nm wasdetermined. OD570 is directly proportional to antiviral activity.

The results show the antiviral activity when IL-29 and IFN on weretested with HepG2 cells: IL-29, IFN-β and IFN α-2a were added at varyingconcentration to HepG2 cells prior to EMCV infection and dye-uptakeassay. The mean and standard deviation of the OD570 from triplicatewells is plotted. OD570 is directly proportional to antiviral activity.For IL-29, the EC50 was 0.60 ng/ml; for IFN-α2a, the EC50 was 0.57ng/ml; and for IFN-β, the EC50 was 0.46 ng/ml.

Example 23 IL-28RA mRNA Expression in Liver and Lymphocyte Subsets

In order to further examine the mRNA distribution for IL-28RA,semi-quantitative RT-PCR was performed using the SDS 7900HT system(Applied Biosystems, Calif.). One-step RT-PCR was performed using 100 ngtotal RNA for each sample and gene-specific primers. A standard curvewas generated for each primer set using Bjab RNA and all sample valueswere normalized to HPRT. The normalized results are summarized in Tables14-17. The normalized values for IFNAR2 and CRF2-4 are also shown. TABLE14 B and T cells express significant levels of IL-28RA mRNA. Low levelsare seen in dendritic cells and most monocytes. Table 14: Cell/TissueIL-28RA IFNAR2 CRF2-4 Dendritic Cells unstim .04 5.9 9.8 DendriticCells + IFNg .07 3.6 4.3 Dendritic Cells .16 7.85 3.9 CD14+ stim'd withLPS/IFNg .13 12 27 CD14+ monocytes resting .12 11 15.4 Hu CD14+ Unact.4.2 TBD TBD Hu CD14+ 1 ug/ml LPS act. 2.3 TBD TBD H. Inflamed tonsil 312.4 9.5 H. B-cells + PMA/Iono 4 & 24 hrs 3.6 1.3 1.4 Hu CD19+ resting6.2 TBD TBD Hu CD19+ 4 hr. PMA/Iono 10.6 TBD TBD Hu CD19+ 24 hr Act.PMA/Iono 3.7 TBD TBD IgD+ B-cells 6.47 13.15 6.42 IgM+ B-cells 9.06 15.42.18 IgD− B-cells 5.66 2.86 6.76 NKCells + PMA/Iono 0 6.7 2.9 Hu CD3+Unactivated 2.1 TBD TBD CD4+ resting .9 8.5 29.1 CD4+ Unstim 18 hrs 1.68.4 13.2 CD4+ + Poly I/C 2.2 4.5 5.1 CD4+ + PMA/Iono .3 1.8 .9 CD3 negresting 1.6 7.3 46 CD3 neg unstim 18 hrs 2.4 13.2 16.8 CD3 neg + PolyI/C 18 hrs 5.7 7 30.2 CD3 neg + LPS 18 hrs 3.1 11.9 28.2 CD8+ unstim 18hrs 1.8 4.9 13.1 CD8+ stim'd with PMA/Ion 18 hrs .3 .6 1.1

As shown in Table 14, normal liver tissue and liver derived cell linesdisplay substantial levels of IL-28RA and CRF2-4 mRNA. TABLE 15Cell/Tissue IL-28RA IFNAR2 CRF2-4 HepG2 1.6 3.56 2.1 HepG2 UGAR 5/10/021.1 1.2 2.7 HepG2, CGAT HKES081501C 4.3 2.1 6 HuH7 5/10/02 1.63 16 2HuH7 hepatoma - CGAT 4.2 7.2 3.1 Liver, normal - CGAT # 11.7 3.2 8.4HXYZ020801K Liver, NAT - Normal adjacent tissue 4.5 4.9 7.7 Liver, NAT -Normal adjacent tissue 2.2 6.3 10.4 Hep SMVC hep vein 0 1.4 6.5 Hep SMCAhep. Artery 0 2.1 7.5 Hep. Fibro 0 2.9 6.2 Hep. Ca. 3.8 2.9 5.8 Adenocaliver 8.3 4.2 10.5 SK-Hep-1 adenoca. Liver .1 1.3 2.5 AsPC-1 Hu.Pancreatic adenocarc. .7 .8 1.3 Hu. Hep. Stellate cells .025 4.4 9.7

As shown in Table 15, primary airway epithelial cells contain abundantlevels of IL-28RA and CRF2-4. TABLE 16 Cell/Tissue IL-28RA IFNAR2 CRF2-4U87MG - glioma 0 .66 .99 NHBE unstim 1.9 1.7 8.8 NHBE + TNF-alpha 2.25.7 4.6 NHBE + poly I/C 1.8 nd nd Small Airway Epithelial Cells 3.9 3.327.8 NHLF—Normal human lung fibroblasts 0 nd nd

As shown in Table 16, IL-28RA is present in normal and diseased liverspecimens, with increased expression in tissue from Hepatitis C andHepatitis B infected specimens. TABLE 17 Cell/Tissue IL-28RA CRF2-4IFNAR2 Liver with Coagulation Necrosis 8.87 15.12 1.72 Liver withAutoimmune Hepatitis 6.46 8.90 3.07 Neonatal Hepatitis 6.29 12.46 6.16Endstage Liver disease 4.79 17.05 10.58 Fulminant Liver Failure 1.9014.20 7.69 Fulminant Liver failure 2.52 11.25 8.84 Cirrhosis, primarybiliary 4.64 12.03 3.62 Cirrhosis Alcoholic (Laennec's) 4.17 8.30 4.14Cirrhosis, Cryptogenic 4.84 7.13 5.06 Hepatitis C+, with cirrhosis 3.647.99 6.62 Hepatitis C+ 6.32 11.29 7.43 Fulminant hepatitis secondary toHep A 8.94 21.63 8.48 Hepatitis C+ 7.69 15.88 8.05 Hepatitis B+ 1.6112.79 6.93 Normal Liver 8.76 5.42 3.78 Normal Liver 1.46 4.13 4.83 LiverNAT 3.61 5.43 6.42 Liver NAT 1.97 10.37 6.31 Hu Fetal Liver 1.07 4.873.98 Hepatocellular Carcinoma 3.58 3.80 3.22 Adenocarcinoma Liver 8.3010.48 4.17 hep. SMVC, hep. Vein 0.00 6.46 1.45 Hep SMCA hep. Artery 0.007.55 2.10 Hep. Fibroblast 0.00 6.20 2.94 HuH7 hepatoma 4.20 3.05 7.24HepG2 Hepatocellular carcinoma 3.40 5.98 2.11 SK-Hep-1 adenocar. Liver0.03 2.53 1.30 HepG2 Unstim 2.06 2.98 2.28 HepG2 + zcyto21 2.28 3.012.53 HepG2 + IFNα 2.61 3.05 3.00 Normal Female Liver - degraded 1.386.45 4.57 Normal Liver - degraded 1.93 4.99 6.25 Normal Liver - degraded2.41 2.32 2.75 Disease Liver - degraded 2.33 3.00 6.04 PrimaryHepatocytes from Clonetics 9.13 7.97 13.30

As shown in Tables 18-22, IL-28RA is detectable in normal B cells, Blymphoma cell lines, T cells, T lymphoma cell lines (Jurkat), normal andtransformed lymphocytes (B cells and T cells) and normal humanmonocytes. TABLE 18 HPRT IL-28RA IL-28RA IFNR2 CRF2-4 Mean Mean normIFNAR2 norm CRF2-4 Norm CD14+ 24 hr unstim #A38 13.1 68.9 5.2 92.3 7.0199.8 15.2 CD14+ 24 hr stim #A38 6.9 7.6 1.1 219.5 31.8 276.6 40.1 CD14+24 hr unstim #A112 17.5 40.6 2.3 163.8 9.4 239.7 13.7 CD14+ 24 hr stim#A112 11.8 6.4 0.5 264.6 22.4 266.9 22.6 CD14+ rest #X 32.0 164.2 5.11279.7 39.9 699.9 21.8 CD14+ + LPS #X 21.4 40.8 1.9 338.2 15.8 518.024.2 CD14+ 24 hr unstim #A39 26.3 86.8 3.3 297.4 11.3 480.6 18.3 CD14+24 hr stim #A39 16.6 12.5 0.8 210.0 12.7 406.4 24.5 HL60 Resting 161.20.2 0.0 214.2 1.3 264.0 1.6 HL60 + PMA 23.6 2.8 0.1 372.5 15.8 397.516.8 U937 Resting 246.7 0.0 0.0 449.4 1.8 362.5 1.5 U937 + PMA 222.7 0.00.0 379.2 1.7 475.9 2.1 Jurkat Resting 241.7 103.0 0.4 327.7 1.4 36.10.1 Jurkat Activated 130.7 143.2 1.1 Colo205 88.8 43.5 0.5 HT-29 26.530.5 1.2

TABLE 19 HPRT SD IL-28RA SD Mono 24 hr unstim #A38 0.6 2.4 Mono 24 hrstim #A38 0.7 0.2 Mono 24 hr unstim #A112 2.0 0.7 Mono 24 hr stim #A1120.3 0.1 Mono rest #X 5.7 2.2 Mono + LPS #X 0.5 1.0 Mono 24 hr unstim#A39 0.7 0.8 Mono 24 hr stim #A39 0.1 0.7 HL60 Resting 19.7 0.1 HL60 +PMA 0.7 0.4 U937 Resting 7.4 0.0 U937 + PMA 7.1 0.0 Jurkat Resting 3.71.1 Jurkat Activated 2.4 1.8 Colo205 1.9 0.7 HT-29 2.3 1.7

TABLE 20 Mean Mean Mean Mean Hprt IFNAR2 IL-28RA CRF CD3+/CD4+ 0 10.185.9 9.0 294.6 CD4/CD3+ Unstim 18 hrs 12.9 108.7 20.3 170.4 CD4+/CD3+ +Poly I/C 18 hrs 24.1 108.5 52.1 121.8 CD4+/CD3+ + PMA/Iono 18 hrs 47.883.7 16.5 40.8 CD3 neg 0 15.4 111.7 24.8 706.1 CD3 neg unstim 18 hrs15.7 206.6 37.5 263.0 CD3 neg + Poly I/C 18 hrs 9.6 67.0 54.7 289.5 CD3neg + LPS 18 hrs 14.5 173.2 44.6 409.3 CD8+ Unstim. 18 hrs 6.1 29.7 11.179.9 CD8+ + PMA/Iono 18 hrs 78.4 47.6 26.1 85.5 12.8.1 - NHBE Unstim47.4 81.1 76.5 415.6 12.8.2 - NHBE + TNF-alpha 42.3 238.8 127.7 193.9SAEC 15.3 49.9 63.6 426.0

TABLE 21 IL-28RA CRF IFNAR2 IL-28RA CRF IFNAR2 Norm Norm Norm SD SD SDCD3+/CD4+ 0 0.9 29.1 8.5 0.1 1.6 0.4 CD4/CD3+ Unstim 18 hrs 1.6 13.2 8.40.2 1.6 1.4 CD4+/CD3+ + Poly I/C 18 hrs 2.2 5.1 4.5 0.1 0.3 0.5CD4+/CD3+ + PMA/Iono 18 hrs 0.3 0.9 1.8 0.0 0.1 0.3 CD3 neg 0 1.6 46.07.3 0.2 4.7 1.3 CD3 neg unstim 18 hrs 2.4 16.8 13.2 0.4 2.7 2.3 CD3neg + Poly I/C 18 hrs 5.7 30.2 7.0 0.3 1.7 0.8 CD3 neg + LPS 18 hrs 3.128.2 11.9 0.4 5.4 2.9 CD8+ Unstim. 18 hrs 1.8 13.1 4.9 0.1 1.1 0.3CD8+ + PMA/Iono 18 hrs 0.3 1.1 0.6 0.0 0.1 0.0 12.8.1 - NHBE Unstim 1.68.8 1.7 0.1 0.4 0.1 12.8.2 - NHBE + TNF-alpha 3.0 4.6 5.7 0.1 0.1 0.1SAEC 4.1 27.8 3.3 0.2 1.1 0.3

TABLE 22 SD SD IL- SD SD Hprt IFNAR2 28RA CRF CD3+/CD4+ 0 0.3 3.5 0.612.8 CD4/CD3+ Unstim 18 hrs 1.4 13.7 1.1 8.5 CD4+/CD3+ + Poly I/C 18 hrs1.3 9.8 1.6 3.4 CD4+/CD3+ + PMA/Iono 18 hrs 4.0 10.3 0.7 3.7 CD3 neg 01.4 16.6 1.6 28.6 CD3 neg unstim 18 hrs 2.4 16.2 2.7 12.6 CD3 neg + PolyI/C 18 hrs 0.5 7.0 1.0 8.3 CD3 neg + LPS 18 hrs 1.0 39.8 5.6 73.6 CD8+Unstim. 18 hrs 0.2 1.6 0.5 6.1 CD8+ + PMA/Iono 18 hrs 1.3 1.7 0.2 8.112.8.1 - NHBE Unstim 2.4 5.6 2.7 2.8 12.8.2 - NHBE + TNF-alpha 0.5 3.43.5 3.4 SAEC 0.5 4.8 1.8 9.9

Example 24 Mouse IL-28 Does Not Effect Daudi Cell Proliferation

Human Daudi cells were suspended in RPMI+10% FBS at 50,000cells/milliliter and 5000 cells were plated per well in a 96 well plate.IL-29-CEE (IL-29 conjugated with glu tag), IFN-γ or IFN-α2a was added in2-fold serial dilutions to each well. IL-29-CEE was used at aconcentration range of from 1000 ng/ml to 0.5 ng/ml. IFN-γ was used at aconcentration range from 125 ng/ml to 0.06 ng/ml. IFN-α2a was used at aconcentration range of from 62 ng/ml to 0.03 ng/ml. Cells were incubatedfor 72 h at 37° C. After 72 hrs Alamar Blue (Accumed, Chicago, Ill.) wasadded at 20 microliters/well. Plates were further incubated at 37° C.,5% CO, for 24 hours. Plates were read on the Fmax™ plate reader(Molecular Devices, Sunnyvale, Calif.) using the SoftMax™ Pro program,at wavelengths 544 (Excitation) and 590 (Emission). Alamar Blue gives afluourometric readout based on the metabolic activity of cells, and isthus a direct measurement of cell proliferation in comparison to anegative control. The results indicate that IL-29-CEE, in contrast toIFN-α2a, has no significant effect on proliferation of Daudi cells.

Example 25 Mouse IL-28 Does Not Have Antiproliferative Effect on Mouse BCells

Mouse B cells were isolated from 2 Balb/C spleens (7 months old) bydepleting CD43+ cells using MACS magnetic beads. Purified B cells werecultured in vitro with LPS, anti-IgM or anti-CD40 monoclonal antibodies.Mouse IL-28 or mouse IFNA was added to the cultures and ³H-thymidine wasadded at 48 hrs. and ³H-thymidine incorporation was measured after 72hrs. culture.

IFNA at 10 ng/ml inhibited ³H-thymidine incorporation by mouse B cellsstimulated with either LPS or anti-IgM. However mouse IL-28 did notinhibit ³H-thymidine incorporation at any concentration tested including1000 ng/ml. In contrast, both mIFNα and mouse IL-28 increased ³Hthymidine incorporation by mouse B cells stimulated with anti-CD40 MAb.

These data demonstrate that mouse IL-28 unlike IFNa displays noantiproliferative activity even at high concentrations. In addition,zcyto24 enhances proliferation in the presence of anti-CD40 MAbs. Theresults illustrate that mouse IL-28 differs from IFNα in that mouseIL-28 does not display antiproliferative activity on mouse B cells, evenat high concentrations. In addition, mouse IL-28 enhances proliferationin the presence of anti-CD40 monoclonal antibodies.

Example 26 Bone Marrow Expansion Assay

Fresh human marrow mononuclear cells (Poietic Technologies,Gaithersburg, Md.) were adhered to plastic for 2 hrs in αMEM, 10% FBS,50 micromolar {tilde over (β)}mercaptoethanol, 2 ng/ml FLT3L at 37° C.Non adherent cells were then plated at 25,000 to 45,000 cells/well (96well tissue culture plates) in αMEM, 10% FBS, 50 micromolar {tilde over(β)}mercaptoethanol, 2 ng/ml FLT3L in the presence or absence of 1000ng/ml IL-29-CEE, 100 ng/ml IL-29-CEE, 10 ng/ml IL-29-CEE, 100 ng/mlIFN-α2a, 10 ng/ml IFN-α2a or 1 ng/ml IFN-α2a. These cells were incubatedwith a variety of cytokines to test for expansion or differentiation ofhematopoietic cells from the marrow (20 ng/ml IL-2, 2 ng/ml IL-3, 20ng/ml IL-4, 20 ng/ml IL-5, 20 ng/ml IL-7, 20 ng/ml IL-10, 20 ng/mlIL-12, 20 ng/ml IL-15, 10 ng/ml IL-21 or no added cytokine). After 8 to12 days Alamar Blue (Accumed, Chicago, Ill.) was added at 20microliters/well. Plates were further incubated at 37° C., 5% CO, for 24hours. Plates were read on the Fmax™ plate reader (Molecular DevicesSunnyvale, Calif.) using the SoftMax™ Pro program, at wavelengths 544(Excitation) and 590 (Emission). Alamar Blue gives a fluourometricreadout based on the metabolic activity of cells, and is thus a directmeasurement of cell proliferation in comparison to a negative control.

IFN-α2a caused a significant inhibition of bone marrow expansion underall conditions tested. In contrast, IL-29 had no significant effect onexpansion of bone marrow cells in the presence of IL-3, IL-4, IL-5,IL-7, IL-10, IL-12, IL-21 or no added cytokine. A small inhibition ofbone marrow cell expansion was seen in the presence of IL-2 or IL-15.

Example 27 Inhibition of IL-28 and IL-29 Signaling with Soluble Receptor(ZcytoR19/CRF2-4)

A. Signal Transduction Reporter Assay

A signal transduction reporter assay can be used to show the inhibitorproperties of zcytor19-Fc4 homodimeric and zcytor19-Fc/CRF2-4-Fcheterodimeric soluble receptors on zcyto20, zcyto21 and zcyto24signaling. Human embryonal kidney (HEK) cells overexpressing thezcytor19 receptor are transfected with a reporter plasmid containing aninterferon-stimulated response element (ISRE) driving transcription of aluciferase reporter gene. Luciferase activity following stimulation oftransfected cells with ligands (including zcyto20 (SEQ ID NO:2), zcyto21(SEQ ID NO:4), zcyto24 (SEQ ID NO:8)) reflects the interaction of theligand with soluble receptor.

B. Cell Transfections

293 HEK cells overexpressing zcytor19 were transfected as follows:700,000 293 cells/well (6 well plates) were plated approximately 18 hprior to transfection in 2 milliliters DMEM+10% fetal bovine serum. Perwell, 1 microgram pISRE-Luciferase DNA (Stratagene) and 1 microgrampIRES2-EGFP DNA (Clontech,) were added to 6 microliters Fugene 6 reagent(Roche Biochemicals) in a total of 100 microliters DMEM. Thistransfection mix was added 30 minutes later to the pre-plated 293 cells.Twenty-four hours later the transfected cells were removed from theplate using trypsin-EDTA and replated at approximately 25,000 cells/wellin 96 well microtiter plates. Approximately 18 h prior to ligandstimulation, media was changed to DMEM+0.5% FBS.

C. Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37° C. in DMEM+0.5% FBS, transfected cells werestimulated with 10 ng/ml zcyto20, zcyto21 or zcyto24 and 10micrograms/ml of the following soluble receptors; human zcytor19-Fchomodimer, human zcytor19-Fc/human CRF2-4-Fc heterodimer, humanCRF2-4-Fc homodimer, murine zcytor19-Ig homodimer. Following a 4-hourincubation at 37° C., the cells were lysed, and the relative light units(RLU) were measured on a luminometer after addition of a luciferasesubstrate. The results obtained are shown as the percent inhibition ofligand-induced signaling in the presence of soluble receptor relative tothe signaling in the presence of PBS alone. Table 23 shows that thehuman zcytor19-Fc/human CRF2-4 heterodimeric soluble receptor is able toinhibit zcyto20, zcyto21 and zcyto24-induced signaling between 16 and45% of control. The human zcytor19-Fc homodimeric soluble receptor isalso able to inhibit zcyto21-induced signaling by 45%. No significanteffects were seen with huCRF2-4-Fc or muzcytor19-Ig homodimeric solublereceptors. TABLE 23 Percent Inhibition of Ligand-induced InterferonStimulated Response Element (ISRE) Signaling by Soluble ReceptorsHuzcytor19- HuCRF2- Ligand Fc/huCRF2-4-Fc Huzcytor19-Fc 4-FcMuzcytor19-Ig Zcyto20 16% 92% 80% 91% Zcyto21 16% 45% 79% 103% Zcyto2447% 90% 82% 89%

Example 28 IL-28 and IL-29 Inhibit HIV Replication in Fresh Human PBMCs

Human immunodeficiency virus (HIV) is a pathogenic retrovirus thatinfects cells of the immune system. CD4 T cells and monocytes are theprimary infected cell types. To test the ability of IL-28 and IL-29 toinhibit HIV replication in vitro, PBMCs from normal donors were infectedwith the HIV virus in the presence of IL-28, IL-29 andMetIL-29C172S-PEG.

Fresh human peripheral blood mononuclear cells (PBMCs) were isolatedfrom whole blood obtained from screened donors who were seronegative forHIV and HBV. Peripheral blood cells were pelleted/washed 2-3 times bylow speed centrifugation and resuspended in PBS to remove contaminatingplatelets. The washed blood cells were diluted 1:1 with Dulbecco'sphosphate buffered saline (D-PBS) and layered over 14 mL of LymphocyteSeparation Medium ((LSM; cellgro™ by Mediatech, Inc. Herndon, Va.);density 1.078±0.002 g/ml) in a 50 mL centrifuge tube and centrifuged for30 minutes at 600×G. Banded PBMCs were gently aspirated from theresulting interface and subsequently washed 2× in PBS by low speedcentrifugation. After the final wash, cells were counted by trypan blueexclusion and resuspended at 1×10⁷ cells/mL in RPMI 1640 supplementedwith 15% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 4 μg/mL PHA-P. Thecells were allowed to incubate for 48-72 hours at 37° C. Afterincubation, PBMCs were centrifuged and resuspended in RPMI 1640 with 15%FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10μg/mL gentamycin, and 20 U/mL recombinant human IL-2. PBMCs weremaintained in the medium at a concentration of 1-2×10⁶ cells/mL withbiweekly medium changes until used in the assay protocol. Monocytes weredepleted from the culture as the result of adherence to the tissueculture flask.

For the standard PBMC assay, PHA-P stimulated cells from at least twonormal donors were pooled, diluted in fresh medium to a finalconcentration of 1×10⁶ cells/mL, and plated in the interior wells of a96 well round bottom microplate at 50 μL/well (5×10⁴ cells/well). Testdilutions were prepared at a 2× concentration in microtiter tubes and100 μL of each concentration was placed in appropriate wells in astandard format. IL-28, IL-29 and MetIL-29C172S-PEG were added atconcentrations from 0-10 μg/ml, usually in ½ log dilutions. 50 μL of apredetermined dilution of virus stock was placed in each test well(final MOI of 0.1). Wells with only cells and virus added were used forvirus control. Separate plates were prepared identically without virusfor drug cytotoxicity studies using an MTS assay system. The PBMCcultures were maintained for seven days following infection, at whichtime cell-free supernatant samples were collected and assayed forreverse transcriptase activity and p24 antigen levels.

A decrease in reverse transcriptase activity or p24 antigen levels withIL-28, IL-29 and MetIL-29C172S-PEG would be indicators of antiviralactivity. Result would demonstrate that IL-28 and IL-29 may havetherapeutic value in treating HIV and AIDS.

Example 29 IL-28 and IL-29 Inhibit GBV-B Replication in Marmoset LiverCells

HCV is a member of the Flaviviridae family of RNA viruses. HCV does notreplicate well in either ex-vivo or in vitro cultures and therefore,there are no satisfactory systems to test the anti-HCV activity ofmolecules in vitro. GB virus B (GBV-B) is an attractive surrogate modelfor use in the development of anti-HCV antiviral agents since it has arelatively high level of sequence identity with HCV and is ahepatotropic virus. To date, the virus can only be grown in the primaryhepatocytes of certain non-human primates. This is accomplished byeither isolating hepatocytes in vitro and infecting them with GBV-B, orby isolating hepatocytes from GBV-B infected marmosets and directlyusing them with antiviral compounds.

The effects of IL-28, IL-29 and MetIL-29C172S-PEG are assayed on GBV-Bextracellular RNA production by TaqMan RT-PCR and on cytotoxicity usingCellTiter96® reagent (Promega, Madison, Wis.) at six half-log dilutionsIL-28, IL-29 or MetIL-29C172S-PEG polypeptide in triplicate. Untreatedcultures serve as the cell and virus controls. Both RIBAVIRIN® (200μg/ml at the highest test concentration) and IFN-α (5000 IU/ml at thehighest test) are included as positive control compounds. Primaryhepatocyte cultures are isolated and plated out on collagen-coatedplates. The next day the cultures are treated with the test samples(IL-28, IL-29, MetIL-29C172S-PEG, IFNα, or RIBAVIRIN®) for 24 hr beforebeing exposed to GBV-B virions or treated directly with test sampleswhen using in vivo infected hepatocytes. Test samples and media areadded the next day, and replaced three days later. Three to four dayslater (at day 6-7 post test sample addition) the supernatant iscollected and the cell numbers quantitated with CellTiter96®. Viral RNAis extracted from the supernatant and quantified with triplicatereplicates in a quantitative TaqMan RT-PCR assay using an in vitrotranscribed RNA containing the RT-PCR target as a standard. The averageof replicate samples is computed. Inhibition of virus production isassessed by plotting the average RNA and cell number values of thetriplicate samples relative to the untreated virus and cell controls.The inhibitory concentration of drug resulting in 50% inhibition ofGBV-B RNA production (IC50) and the toxic concentration resulting indestruction of 50% of cell numbers relative to control values (TC50) arecalculated by interpolation from graphs created with the data.

Inhibition of the GBV-B RNA production by IL-28 and 29 is an indicationof the antiviral properties of IL-28 and IL-29 on this Hepatitis C-likevirus on hepatocytes, the primary organ of infection of Hepatitis C, andpositive results suggest that IL-28 or IL-29 may be useful in treatingHCV infections in humans.

Example 30 IL-28, IL-29 and MetIL-29C172S-PEG Inhibit HBV Replication inWT10 Cells

Chronic hepatitis B (HBV) is one of the most common and severe viralinfections of humans belonging to the Hepadnaviridae family of viruses.To test the antiviral activities of IL-28 and IL-29 against HBV, IL-28,IL-29 and MetIL-29C172S-PEG were tested against HBV in an in vitroinfection system using a variant of the human liver line HepG2. IL-28,IL-29 and MetIL-29C172S-PEG inhibited viral replication in this system,suggesting therapeutic value in treating HBV in humans.

WT10 cells are a derivative of the human liver cell line HepG2 2.2.15.WT10 cells are stably transfected with the HBV genome, enabling stableexpression of HBV transcripts in the cell line (Fu and Cheng,Antimicrobial Agents Chemother. 44(12):3402-3407, 2000). In the WT10assay the drug in question and a 3TC control will be assayed at fiveconcentrations each, diluted in a half-log series. The endpoints areTaqMan PCR for extracellular HBV DNA (IC50) and cell numbers usingCellTiter96 reagent (TC50). The assay is similar to that described byKorba et al. Antiviral Res. 15(3):217-228, 1991 and Korba et al.,Antiviral Res. 19(1):55-70, 1992. Briefly, WT10 cells are plated in96-well microtiter plates. After 16-24 hours the confluent monolayer ofHepG2-2.2.15 cells is washed and the medium is replaced with completemedium containing varying concentrations of a test samples intriplicate. 3TC is used as the positive control, while media alone isadded to cells as a negative control (virus control, VC). Three dayslater the culture medium is replaced with fresh medium containing theappropriately diluted test samples. Six days following the initialaddition of the test compound, the cell culture supernatant iscollected, treated with pronase and DNAse, and used in a real-timequantitative TaqMan PCR assay. The PCR-amplified HBV DNA is detected inreal-time by monitoring increases in fluorescence signals that resultfrom the exonucleolytic degradation of a quenched fluorescent probemolecule that hybridizes to the amplified HBV DNA. For each PCRamplification, a standard curve is simultaneously generated usingdilutions of purified HBV DNA. Antiviral activity is calculated from thereduction in HBV DNA levels (IC₅₀). A dye uptake assay is then employedto measure cell viability which is used to calculate toxicity (TC₅₀).The therapeutic index (TI) is calculated as TC₅₀/IC₅₀.

IL-28, IL-29 and MetIL-29C172S-PEG inhibited HepB viral replication inWT10 cells with an IC50<0.032 ug/ml. This demonstrates antiviralactivity of IL-28 and IL-29 against HBV grown in liver cell lines,providing evidence of therapeutic value for treating HBV in humanpatients.

Example 31 IL-28, IL-29 and MetIL-29C172S-PEG Inhibit BVDV Replicationin Bovine Kidney Cells

HCV is a member of the Flaviviridae family of RNA viruses. Other virusesbelonging to this family are the bovine viral diarrhea virus (BVDV) andyellow fever virus (YFV). HCV does not replicate well in either ex vivoor in vitro cultures and therefore there are no systems to test anti-HCVactivity in vitro. The BVDV and YFV assays are used as surrogate virusesfor HCV to test the antiviral activities against the Flavivirida familyof viruses.

The antiviral effects of IL-28, IL-29 and MetIL-29C172S-PEG wereassessed in inhibition of cytopathic effect assays (CPE). The assaymeasured cell death using Madin-Darby bovine kidney cells (MDBK) afterinfection with cytopathic BVDV virus and the inhibition of cell death byaddition of IL-28, IL-29 and MetIL-29C172S-PEG. The MDBK cells werepropagated in Dulbecco's modified essential medium (DMEM) containingphenol red with 10% horse serum, 1% glutamine and 1%penicillin-streptomycin. CPE inhibition assays were performed in DMEMwithout phenol red with 2% FBS, 1% glutamine and 1% Pen-Strep. On theday preceding the assays, cells were trypsinized (1% trypsin-EDTA),washed, counted and plated out at 10⁴ cells/well in a 96-wellflat-bottom BioCoat® plates (Fisher Scientific, Pittsburgh, Pa.) in avolume of 100 μl/well. The next day, the medium was removed and apre-titered aliquot of virus was added to the cells. The amount of viruswas the maximum dilution that would yield complete cell killing (>80%)at the time of maximal CPE development (day 7 for BVDV). Cell viabilitywas determined using a CellTiter96® reagent (Promega) according to themanufacturer's protocol, using a Vmax plate reader (Molecular Devices,Sunnyvale, Calif.). Test samples were tested at six concentrations each,diluted in assay medium in a half-log series. IFNα and RIBAVIRIN® wereused as positive controls. Test sample were added at the time of viralinfection. The average background and sample color-corrected data forpercent CPE reduction and percent cell viability at each concentrationwere determined relative to controls and the IC₅₀ calculated relative tothe TC₅₀.

IL-28, IL-29 and MetIL-29C172S-PEG inhibited cell death induced by BVDVin MDBK bovine kidney cells. IL-28 inhibited cell death with an IC₅₀ of0.02 μg/ml, IL-29 inhibited cell death with an IC₅₀ of 0.19 μg/ml, andMetIL-29C172S-PEG inhibited cell death with an IC₅₀ of 0.45 μg/ml. Thisdemonstrated that IL-28 and IL-29 have antiviral activity against theFlavivirida family of viruses.

Example 32 Induction of Interferon Stimulated Genes by IL-28 and IL-29

A. Human Peripheral Blood Mononuclear Cells

Freshly isolated human peripheral blood mononuclear cells were grown inthe presence of IL-29 (20 ng/mL), IFNα2a (2 ng/ml) (PBL Biomedical Labs,Piscataway, N.J.), or in medium alone. Cells were incubated for 6, 24,48, or 72 hours, and then total RNA was isolated and treated withRNase-free DNase. 100 ng total RNA was used as a template for One-StepSemi-Quantitative RT-PCR® using Taqman One-Step RT-PCR Master Mix®Reagents and gene specific primers as suggested by the manufacturer.(Applied Biosystems, Branchburg, N.J.) Results were normalized to HPRTand are shown as the fold induction over the medium alone control foreach time-point. Table 24 shows that IL-29 induces Interferon StimulatedGene Expression in human peripheral blood mononuclear cells at alltime-points tested. TABLE 24 MxA Fold Pkr Fold OAS Fold inductionInduction Induction  6 hr IL29 3.1 2.1 2.5  6 hr IFNα2a 17.2 9.6 16.2 24hr IL29 19.2 5.0 8.8 24 hr IFNα2a 57.2 9.4 22.3 48 hr IL29 7.9 3.5 3.348 hr IFNα2a 18.1 5.0 17.3 72 hr IL29 9.4 3.7 9.6 72 hr IFNα2a 29.9 6.447.3B. Activated Human T Cells

Human T cells were isolated by negative selection from freshly harvestedperipheral blood mononuclear cells using the Pan T-cell Isolation® kitaccording to manufacturer's instructions (Miltenyi, Auburn, Calif.). Tcells were then activated and expanded for 5 days with plate-boundanti-CD3, soluble anti-CD28 (0.5 ug/ml), (Pharmingen, San Diego, Calif.)and Interleukin 2 (IL-2; 100 U/ml) (R&D Systems, Minneapolis, Minn.),washed and then expanded for a further 5 days with IL-2. Followingactivation and expansion, cells were stimulated with IL-28A (20 ng/ml),IL-29 (20 ng/ml), or medium alone for 3, 6, or 18 hours. Total RNA wasisolated and treated with RNase-Free DNase. One-Step Semi-QuantitativeRT-PCR® was performed as described in the example above. Results werenormalized to HPRT and are shown as the fold induction over the mediumalone control for each time-point. Table 25 shows that IL-28 and IL-29induce Interferon Stimulated Gene expression in activated human T cellsat all time-points tested. TABLE 25 MxA Fold Pkr Fold OAS Fold InductionInduction Induction Donor #1 3 hr IL28 5.2 2.8 4.8 Donor #1 3 hr IL295.0 3.5 6.0 Donor #1 6 hr IL28 5.5 2.2 3.0 Donor #1 6 hr IL29 6.4 2.23.7 Donor #1 18 hr IL28 4.6 4.8 4.0 Donor #1 18 hr IL29 5.0 3.8 4.1Donor #2 3 hr IL28 5.7 2.2 3.5 Donor #2 3 hr IL29 6.2 2.8 4.7 Donor #2 6hr IL28 7.3 1.9 4.4 Donor #2 6 hr IL29 8.7 2.6 4.9 Donor #2 18 hr IL284.7 2.3 3.6 Donor #2 18 hr IL29 4.9 2.1 3.8C. Primary Human Hepatocytes

Freshly isolated human hepatocytes from two separate donors (Cambrex,Baltimore, Md. and CellzDirect, Tucson, Ariz.) were stimulated withIL-28A (50 ng/ml), IL-29 (50 ng/ml), IFNα2a (50 ng/ml), or medium alonefor 24 hours. Following stimulation, total RNA was isolated and treatedwith RNase-Free DNase. One-step semi-quantitative RT-PCR was performedas described previously in the example above. Results were normalized toHPRT and are shown as the fold induction over the medium alone controlfor each time-point. Table 26 shows that IL-28 and IL-29 induceInterferon Stimulated Gene expression in primary human hepatocytesfollowing 24-hour stimulation. TABLE 26 MxA Fold Pkr Fold OAS FoldInduction Induction Induction Donor #1 IL28 31.4 6.4 30.4 Donor #1 IL2931.8 5.2 27.8 Donor #1 IFN-α2a 63.4 8.2 66.7 Donor #2 IL28 41.7 4.2 24.3Donor #2 IL29 44.8 5.2 25.2 Donor #2 IFN-α2a 53.2 4.8 38.3D. HepG2 and HuH7: Human Liver Hepatoma Cell Lines

HepG2 and HuH7 cells (ATCC NOS. 8065, Manassas, Va.) were stimulatedwith IL-28A (10 ng/ml), IL-29 (10 ng/ml), IFNα2a (10 ng/ml), IFNB (1ng/ml) (PBL Biomedical, Piscataway, N.J.), or medium alone for 24 or 48hours. In a separate culture, HepG2 cells were stimulated as describedabove with 20 ng/ml of MetIL-29C172S-PEG or MetIL-29-PEG. Total RNA wasisolated and treated with RNase-Free DNase. 100 ng Total RNA was used asa template for one-step semi-quantitative RT-PCR as describedpreviously. Results were normalized to HPRT and are shown as the foldinduction over the medium alone control for each time-point. Table 27shows that IL-28 and IL-29 induce ISG expression in HepG2 and HuH7 liverhepatoma cell lines after 24 and 48 hours. TABLE 27 MxA Fold Pkr FoldOAS Fold Induction Induction Induction HepG2 24 hr IL28 12.4 0.7 3.3HepG2 24 hr IL29 36.6 2.2 6.4 HepG2 24 hr IFNα2a 12.2 1.9 3.2 HepG2 24hr IFNβ 93.6 3.9 19.0 HepG2 48 hr IL28 2.7 0.9 1.1 HepG2 48 hr IL29 27.22.1 5.3 HepG2 48 hr IFNα2a 2.5 0.9 1.2 HepG2 48 hr IFNβ 15.9 1.8 3.3HuH7 24 hr IL28 132.5 5.4 52.6 HuH7 24 hr IL29 220.2 7.0 116.6 HuH7 24hr IFNα2a 157.0 5.7 67.0 HuH7 24 hr IFNβ 279.8 5.6 151.8 HuH7 48 hr IL2825.6 3.4 10.3 HuH7 48 hr IL29 143.5 7.4 60.3 HuH7 48 hr IFNα2a 91.3 5.832.3 HuH7 48 hr IFNβ 65.0 4.2 35.7

TABLE 28 MxA Fold OAS Fold Pkr Fold Induction Induction InductionMetIL-29-PEG 36.7 6.9 2.2 MetIL-29C172S-PEG 46.1 8.9 2.8

Data shown is for 20 ng/ml metIL-29-PEG and metIL-29C172S-PEG versionsof IL-29 after culture for 24 hours.

Data shown is normalized to HPRT and shown as fold induction overunstimulated cells.

Example 33 Antiviral Activity of IL-28 and IL-29 in HCV Replicon System

The ability of antiviral drugs to inhibit HCV replication can be testedin vitro with the HCV replicon system. The replicon system consists ofthe Huh7 human hepatoma cell line that has been transfected withsubgenomic RNA replicons that direct constitutive replication of HCVgenomic RNAs (Blight, K. J. et al. Science 290:1972-1974, 2000).Treatment of replicon clones with IFNα at 10 IU/ml reduces the amount ofHCV RNA by 85% compared to untreated control cell lines. The ability ofIL-28A and IL-29 to reduce the amount of HCV RNA produced by thereplicon clones in 72 hours indicates the antiviral state conferred uponHuh7 cells by IL-28A/IL-29 treatment is effective in inhibiting HCVreplicon replication, and thereby, very likely effective in inhibitingHCV replication.

The ability of IL-28A and IL-29 to inhibit HCV replication as determinedby Bayer Branched chain DNA kit, is be done under the followingconditions:

IL28 alone at increasing concentrations (6)* up to 1.0 μg/ml

IL29 alone at increasing concentrations (6)* up to 1.0 μg/ml

PEGIL29 alone at increasing concentrations (6)* up to 1.0 μg/ml

IFNα2A alone at 0.3, 1.0, and 3.0 IU/ml

Ribavirin alone.

The positive control is IFNα and the negative control is ribavirin.

The cells are stained after 72 hours with Alomar Blue to assessviablility.

*The concentrations for conditions 1-3 are:

μg/ml, 0.32 μg/ml, 0.10 μg/ml, 0.032 μg/ml, 0.010 μg/ml, 0.0032 μg/ml.

The replicon clone (BB7) is treated 1× per day for 3 consecutive dayswith the doses listed above. Total HCV RNA is measured after 72 hours.

Example 34 IL-28 and IL-29 have Antiviral Activity Against PathogenicViruses

Two methods are used to assay in vitro antiviral activity of IL-28 andIL-29 against a panel of pathogenic viruses including, among others,adenovirus, parainfluenza virus. respiratory syncytial virus, rhinovirus, coxsackie virus, influenza virus, vaccinia virus, west nilevirus, dengue virus, venezuelan equine encephalitis virus, pichindevirus and polio virus. These two methods are inhibition of virus-inducedcytopathic effect (CPE) determined by visual (microscopic) examinationof the cells and increase in neutral red (NR) dye uptake into cells. Inthe CPE inhibition method, seven concentrations of test drug (log10dilutions, such as 1000, 100, 10, 1, 0.1, 0.01, 0.001 ng/ml) areevaluated against each virus in 96-well flat-bottomed microplatescontaining host cells. The compounds are added 24 hours prior to virus,which is used at a concentration of approximately 5 to 100 cell cultureinfectious doses per well, depending upon the virus, which equates to amultiplicity of infection (MOI) of 0.01 to 0.0001 infectious particlesper cell. The tests are read after incubation at 37° C. for a specifiedtime sufficient to allow adequate viral cytopathic effect to develop. Inthe NR uptake assay, dye (0.34% concentration in medium) is added to thesame set of plates used to obtain the visual scores. After 2 h, thecolor intensity of the dye absorbed by and subsequently eluted from thecells is determined using a microplate autoreader. Antiviral activity isexpressed as the 50% effective (virus-inhibitory) concentration (EC50)determined by plotting compound concentration versus percent inhibitionon semilogarithmic graph paper. The EC50/IC50 data in some cases may bedetermined by appropriate regression analysis software. In general, theEC50s determined by NR assay are two-to fourfold higher than thoseobtained by the CPE method. TABLE 29 Visual Assay SI Visual Virus Cellline Drug EC50 Visual IC50 Visual (IC50/EC50) Adenovirus A549 IL-28A >10μg/ml >10 μg/ml 0 Adenovirus A549 IL-29 >10 μg/ml >10 μg/ml 0 AdenovirusA549 MetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEG Parainfluenza MA-104IL-28A >10 μg/ml >10 μg/ml 0 virus Parainfluenza MA-104 IL-29 >10μg/ml >10 μg/ml 0 virus Parainfluenza MA-104 MetIL-29C172S- >10μg/ml >10 μg/ml 0 virus PEG Respiratory MA-104 IL-28A >10 μg/ml >10μg/ml 0 syncytial virus Respiratory MA-104 IL-29 >10 μg/ml >10 μg/ml 0syncytial virus Respiratory MA-104 MetIL-29C172S- >10 μg/ml >10 μg/ml 0syncytial virus PEG Rhino 2 KB IL-28A >10 μg/ml >10 μg/ml 0 Rhino 2 KBIL-29 >10 μg/ml >10 μg/ml 0 Rhino 2 KB MetIL-29C172S- >10 μg/ml >10μg/ml 0 PEG Rhino 9 HeLa IL-28A >10 μg/ml >10 μg/ml 0 Rhino 9 HeLaIL-29 >10 μg/ml >10 μg/ml 0 Rhino 9 HeLa MetIL-29C172S- >10 μg/ml >10μg/ml 0 PEG Coxsackie B4 KB IL-28A >10 μg/ml >10 μg/ml 0 virus CoxsackieB4 KB IL-29 >10 μg/ml >10 μg/ml 0 virus Coxsackie B4 KBMetIL-29C172S- >10 μg/ml >10 μg/ml 0 virus PEG Influenza (type Maden-IL-28A >10 μg/ml >10 μg/ml 0 A [H3N2]) Darby Canine Kidney Influenza(type Maden- IL-29 >10 μg/ml >10 μg/ml 0 A [H3N2]) Darby Canine KidneyInfluenza (type Maden- MetIL-29C172S- >10 μg/ml >10 μg/ml 0 A [H3N2])Darby PEG Canine Kidney Influenza (type Vero IL-28A  0.1 μg/ml >10μg/ml >100 A [H3N2]) Influenza (type Vero IL-29 >10 μg/ml >10 μg/ml 0 A[H3N2]) Influenza (type Vero MetIL-29C172S- 0.045 μg/ml   >10 μg/ml >222A [H3N2]) PEG Vaccinia virus Vero IL-28A >10 μg/ml >10 μg/ml 0 Vacciniavirus Vero IL-29 >10 μg/ml >10 μg/ml 0 Vaccinia virus VeroMetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEG West Nile Vero IL-28A 0.00001μg/ml    >10 μg/ml >1,000,000 virus West Nile Vero IL-29 0.000032μg/ml    >10 μg/ml >300,000 virus West Nile Vero MetIL-29C172S- 0.001μg/ml   >10 μg/ml >10,000 virus PEG Dengue virus Vero IL-28A 0.01μg/ml  >10 μg/ml >1000 Dengue virus Vero IL-29 0.032 μg/ml   >10μg/ml >312 Dengue virus Vero MetIL-29C172S- 0.0075 μg/ml   >10μg/ml >1330 PEG Venezuelan Vero IL-28A 0.01 μg/ml  >10 μg/ml >1000equine encephalitis virus Venezuelan Vero IL-29 0.012 μg/ml   >10μg/ml >833 equine encephalitis virus Venezuelan Vero MetIL-29C172S-0.0065 μg/ml   >10 μg/ml >1538 equine PEG encephalitis virus Pichindevirus BSC-1 IL-28A >10 μg/ml >10 μg/ml 0 Pichinde virus BSC-1 IL-29 >10μg/ml >10 μg/ml 0 Pichinde virus BSC-1 MetIL-29C172S- >10 μg/ml >10μg/ml 0 PEG Polio virus Vero IL-28A >10 μg/ml >10 μg/ml 0 Polio virusVero IL-29 >10 μg/ml >10 μg/ml 0 Polio virus Vero MetIL-29C172S- >10μg/ml >10 μg/ml 0 PEG

TABLE 30 Neutral Red Assay SI NR Virus Cell line Drug EC50 NR IC50 NR(IC50/EC50) Adenovirus A549 IL-28A >10 μg/ml >10 μg/ml 0 Adenovirus A549IL-29 >10 μg/ml >10 μg/ml 0 Adenovirus A549 MetIL-29C172S- >10 μg/ml >10μg/ml 0 PEG Parainfluenza MA-104 IL-28A >10 μg/ml >10 μg/ml 0 virusParainfluenza MA-104 IL-29 >10 μg/ml >10 μg/ml 0 virus ParainfluenzaMA-104 MetIL-29C172S- >10 μg/ml >10 μg/ml 0 virus PEG Respiratory MA-104IL-28A >10 μg/ml >10 μg/ml 0 syncytial virus Respiratory MA-104IL-29 >10 μg/ml >10 μg/ml 0 syncytial virus Respiratory MA-104MetIL-29C172S- 5.47 μg/ml  >10 μg/ml >2 syncytial virus PEG Rhino 2 KBIL-28A >10 μg/ml >10 μg/ml 0 Rhino 2 KB IL-29 >10 μg/ml >10 μg/ml 0Rhino 2 KB MetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEG Rhino 9 HeLa IL-28A1.726 μg/ml   >10 μg/ml >6 Rhino 9 HeLa IL-29 0.982 μg/ml   >10μg/ml >10 Rhino 9 HeLa MetIL-29C172S- 2.051 μg/ml   >10 μg/ml >5 PEGCoxsackie B4 KB IL-28A >10 μg/ml >10 μg/ml 0 virus Coxsackie B4 KBIL-29 >10 μg/ml >10 μg/ml 0 virus Coxsackie B4 KB MetIL-29C172S- >10μg/ml >10 μg/ml 0 virus PEG Influenza (type A Maden- IL-28A >10μg/ml >10 μg/ml 0 [H3N2]) Darby Canine Kidney Influenza (type A Maden-IL-29 >10 μg/ml >10 μg/ml 0 [H3N2]) Darby Canine Kidney Influenza (typeA Maden- MetIL-29C172S- >10 μg/ml >10 μg/ml 0 [H3N2]) Darby PEG CanineKidney Influenza (type A Vero IL-28A 0.25 μg/ml  >10 μg/ml >40 [H3N2])Influenza (type A Vero IL-29  2 μg/ml >10 μg/ml >5 [H3N2]) Influenza(type A Vero MetIL-29C172S-  1.4 μg/ml >10 μg/ml >7 [H3N2]) PEG Vacciniavirus Vero IL-28A >10 μg/ml >10 μg/ml 0 Vaccinia virus Vero IL-29 >10μg/ml >10 μg/ml 0 Vaccinia virus Vero MetIL-29C172S- >10 μg/ml >10 μg/ml0 PEG West Nile virus Vero IL-28A 0.0001 μg/ml   >10 μg/ml >100,000 WestNile virus Vero IL-29 0.00025 μg/ml    >10 μg/ml >40,000 West Nile virusVero MetIL-29C172S- 0.00037 μg/ml    >10 μg/ml >27,000 PEG Dengue virusVero IL-28A  0.1 μg/ml >10 μg/ml >100 Dengue virus Vero IL-29 0.05μg/ml  >10 μg/ml >200 Dengue virus Vero MetIL-29C172S- 0.06 μg/ml  >10μg/ml >166 PEG Venezuelan Vero IL-28A 0.035 μg/ml   >10 μg/ml >286equine encephalitis virus Venezuelan Vero IL-29 0.05 μg/ml  >10μg/ml >200 equine encephalitis virus Venezuelan Vero MetIL-29C172S- 0.02μg/ml  >10 μg/ml >500 equine PEG encephalitis virus Pichinde virus BSC-1IL-28A >10 μg/ml >10 μg/ml 0 Pichinde virus BSC-1 IL-29 >10 μg/ml >10μg/ml 0 Pichinde virus BSC-1 MetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEGPolio virus Vero IL-28A >1.672 μg/ml   >10 μg/ml >6 Polio virus VeroIL-29 >10 μg/ml >10 μg/ml 0 Polio virus Vero MetIL-29C172S- >10μg/ml >10 μg/ml 0 PEG

Example 35 IL-28, IL-29, MetIL-29-PEG and MetIL-29C172S-PEG StimulateISG Induction in the Mouse Liver Cell Line AML-12

Interferon stimulated genes (ISGs) are genes that are induced by type Iinterferons (IFNs) and also by the IL-28 and IL-29 family molecules,suggesting that IFN and IL-28 and IL-29 induce similar pathways leadingto antiviral activity. Human type I IFNs (IFNα1-4 and IFNβ) have littleor no activity on mouse cells, which is thought to be caused by lack ofspecies cross-reactivity. To test if human IL-28 and IL-29 have effectson mouse cells, ISG induction by human IL-28 and IL-29 was evaluated byreal-time PCR on the mouse liver derived cell line AML-12.

AML-12 cells were plated in 6-well plates in complete DMEM media at aconcentration of 2×10⁶ cells/well. Twenty-four hours after platingcells, human IL-28 and IL-29 were added to the culture at aconcentration of 20 ng/ml. As a control, cells were either stimulatedwith mouse IFNα (positive control) or unstimulated (negative). Cellswere harvested at 8, 24, 48 and 72 hours after addition of CHO-derivedhuman IL-28A (SEQ ID NO:2) or IL-29 (SEQ ID NO:4). RNA was isolated fromcell pellets using RNAEasy-kit® (Qiagen, Valencia, Calif.). RNA wastreated with DNase (Millipore, Billerica, Mass.) to clean RNA of anycontaminating DNA. cDNA was generated using Perkin-Elmer RT mix. ISGgene induction was evaluated by real-time PCR using primers and probesspecific for mouse OAS, Pkr and Mx1. To obtain quantitative data, HPRTreal-time PCR was duplexed with ISG PCR. A standard curve was obtainedusing known amounts of RNA from IFN-stimulated mouse PBLs. All data areshown as expression relative to internal HPRT expression.

Human IL-28A and IL-29 stimulated ISG induction in the mouse hepatocytecell line AML-12 and demonstrated that unlike type I IFNs, the IL-28/29family proteins showed cross-species reactivity. TABLE 31 StimulationOAS PkR Mx1 None 0.001 0.001 0.001 Human IL-28 0.04 0.02 0.06 HumanIL-29 0.04 0.02 0.07 Mouse IL-28 0.04 0.02 0.08 Mouse IFNα 0.02 0.020.01

All data shown were expressed as fold relative to HPRT gene expressionng of OAS mRNA=normalized value of OAS mRNA amount relative to internal

ng of HPRT mRNA housekeeping gene, HPRT

As an example, the data for the 48 hour time point is shown. TABLE 32AML12's Mx1 Fold OAS Fold Pkr Fold Induction Induction InductionMetIL-29-PEG 728 614 8 MetIL-29C172S-PEG 761 657 8

Cells were stimulated with 20 ng/ml metIL-29-PEG or metIL-29C172S-PEGfor 24 hours.

Data shown is normalized to HPRT and shown as fold induction overunstimulated cells.

Example 36 ISGs are Efficiently Induced in Spleens of Transzenic MiceExpressing Human IL-29

Transgenic (Tg) mice were generated expressing human IL-29 under thecontrol of the Eu-1ck promoter. To study if human IL-29 has biologicalactivity in vivo in mice, expression of ISGs was analyzed by real-timePCR in the spleens of Eu-1ck IL-29 transgenic mice.

Transgenic mice (C3H/C57BL/6) were generated using a construct thatexpressed the human IL-29 gene under the control of the Eu-1ck promoter.This promoter is active in T cells and B cells. Transgenic mice andtheir non-transgenic littermates (n=2/gp) were sacrificed at about 10weeks of age. Spleens of mice were isolated. RNA was isolated from cellpellets using RNAEasy-kit® (Qiagen). RNA was treated with DNase to cleanRNA of any contaminating DNA. cDNA was generated using Perkin-Elmer RT®mix. ISG gene induction was evaluated by real-time PCR using primers andprobes (5′ FAM, 3′ NFQ) specific for mouse OAS, Pkr and Mx1. To obtainquantitative data, HPRT real-time PCR was duplexed with ISG PCR.Furthermore, a standard curve was obtained using known amounts of IFNstimulated mouse PBLs. All data are shown as expression relative tointernal HPRT expression.

Spleens isolated from IL-29 Tg mice showed high induction of ISGs OAS,Pkr and Mx1 compared to their non-Tg littermate controls suggesting thathuman IL-29 is biologically active in vivo in mice. TABLE 33 Mice OASPkR Mx1 Non-Tg 4.5 4.5 3.5 IL-29 Tg 12 8 21

All data shown are fold expression relative to HPRT gene expression. Theaverage expression in two mice is shown

Example 37 Human IL-28 and IL-29 Protein Induce ISG Gene Expression InLiver, Spleen and Blood of Mice

To determine whether human IL-28 and IL-29 induce interferon stimulatedgenes in vivo, CHO-derived human IL-28A and IL-29 protein were injectedinto mice. In addition, E. coli derived IL-29 was also tested in in vivoassays as described above using MetIL-29C172S-PEG and MetIL-29-PEG. Atvarious time points and at different doses, ISG gene induction wasmeasured in the blood, spleen and livers of the mice.

C57BL/6 mice were injected i.p or i.v with a range of doses (10 μg -250μg) of CHO-derived human IL-28A and IL-29 or MetIL-29C172S-PEG andMetIL-29C16-C113-PEG. Mice were sacrificed at various time points (1hr-48 hr). Spleens and livers were isolated from mice, and RNA wasisolated. RNA was also isolated from the blood cells. The cells werepelleted and RNA isolated from pellets using RNAEasy®-kit (Qiagen). RNAwas treated with DNase (Amicon) to rid RNA of any contaminating DNA.cDNA was generated using Perkin-Elmer RT mix (Perkin-Elmer). ISG geneinduction was measured by real-time PCR using primers and probesspecific for mouse OAS, Pkr and Mx1. To obtain quantitative data, HPRTreal-time PCR was duplexed with ISG PCR. A standard curve was calculatedusing known amounts of IFN-stimulated mouse PBLs. All data are shown asexpression relative to internal HPRT expression.

Human IL-29 induced ISG gene expression (OAS, Pkr, Mx1) in the livers,spleen and blood of mice in a dose dependent manner. Expression of ISGspeaked between 1-6 hours after injection and showed sustained expressionabove control mice upto 48 hours. In this experiment, human IL-28A didnot induce ISG gene expression. TABLE 34 Injection OAS-1 hr OAS-6 hrOAS-24 hr OAS-48 hr None - liver 1.6 1.6 1.6 1.6 IL-29 liver 2.5 4 2.52.8 None - spleen 1.8 1.8 1.8 1.8 IL-29 - spleen 4 6 3.2 3.2 None -blood 5 5 5 5 IL-29 blood 12 18 11 10

Results shown are fold expression relative to HPRT gene expression. Asample data set for IL-29 induced OAS in liver at a single injection of250 μg i.v. is shown. The data shown is the average expression from 5different animals/group. TABLE 35 Injection OAS (24 hr) None 1.8 IL-2910 μg 3.7 IL-29 50 μg 4.2 IL-29 250 μg 6

TABLE 36 MetIL-29-PEG MetIL-29C172S-PEG Naive 3 hr 6 hr 12 hr 24 hr 3 hr6 hr 12 hr 24 hr 24 hr PKR 18.24 13.93 4.99 3.77 5.29 5.65 3.79 3.553.70 OAS 91.29 65.93 54.04 20.81 13.42 13.02 10.54 8.72 6.60 Mx1 537.51124.99 33.58 35.82 27.89 29.34 16.61 0.00 10.98

Mice were injected with 100 μg of proteins i.v. Data shown is foldexpression over HPRT expression from livers of mice. Similar data wasobtained from blood and spleens of mice.

Example 38 IL-28 and IL-29 Induce ISG Protein in Mice

To analyze of the effect of human IL-28 and IL-29 on induction of ISGprotein (OAS), serum and plasma from IL-28 and IL-29 treated mice weretested for OAS activity.

C57BL/6 mice were injected i.v with PBS or a range of concentrations (10μg-250 μg) of human IL-28 or IL-29. Serum and plasma were isolated frommice at varying time points, and OAS activity was measured using the OASradioimmunoassay (RIA) kit from Eiken Chemicals (Tokyo, Japan).

IL-28 and IL-29 induced OAS activity in the serum and plasma of miceshowing that these proteins are biologically active in vivo. TABLE 37Injection OAS-1 hr OAS-6 hr OAS-24 hr OAS-48 hr None 80 80 80 80 IL-2980 80 180 200

OAS activity is shown at pmol/dL of plasma for a single concentration(250 μg) of human IL-29.

Example 39 IL-28 and IL-29 Inhibit Adenoviral Pathology in Mice

To test the antiviral activities of IL-28 and IL-29 against viruses thatinfect the liver, the test samples were tested in mice againstinfectious adenoviral vectors expressing an internal green fluorescentprotein (GFP) gene. When injected intravenously, these viruses primarilytarget the liver for gene expression. The adenoviruses are replicationdeficient, but cause liver damage due to inflammatory cell infiltratethat can be monitored by measurement of serum levels of liver enzymeslike AST and ALT, or by direct examination of liver pathology.

C57B1/6 mice were given once daily intraperitoneal injections of 50 μgmouse IL-28 (zcyto24 as shown in SEQ ID NO:8) or metIL-29C172S-PEG for 3days. Control animals were injected with PBS. One hour following the3^(rd) dose, mice were given a single bolus intravenous tail veininjection of the adenoviral vector, AdGFP (1×10⁹ plaque-forming units(pfu)). Following this, every other day mice were given an additionaldose of PBS, mouse IL-28 or metIL-29C172S-PEG for 4 more doses (total of7 doses). One hour following the final dose of PBS, mouse IL-28 ormetIL-29C172S-PEG mice were terminally bleed and sacrificed. The serumand liver tissue were analyzed. Serum was analyzed for AST and ALT liverenzymes. Liver was isolated and analyzed for GFP expression andhistology. For histology, liver specimens were fixed in formalin andthen embedded in paraffin followed by H&E staining. Sections of liverthat had been blinded to treat were examined with a light microscope.Changes were noted and scored on a scale designed to measure liverpathology and inflammation.

Mouse IL-28 and IL-29 inhibited adenoviral infection and gene expressionas measured by liver fluorescence. PBS-treated mice (n=8) had an averagerelative liver fluorescence of 52.4 (arbitrary units). In contrast,IL-28-treated mice (n=8) had a relative liver fluorescence of 34.5, andIL-29-treated mice (n=8) had a relative liver fluorescence of 38.9. Areduction in adenoviral infection and gene expression led to a reducedliver pathology as measured by serum ALT and AST levels and histology.PBS-treated mice (n=8) had an average serum AST of 234 U/L (units/liter)and serum ALT of 250 U/L. In contrast, IL-28-treated mice (n=8) had anaverage serum AST of 193 U/L and serum ALT of 216 U/L, and IL-29-treatedmice (n=8) had an average serum AST of 162 U/L and serum ALT of 184 U/L.In addition, the liver histology indicated that mice given either mouseIL-28 or IL-29 had lower liver and inflammation scores than thePBS-treated group. The livers from the IL-29 group also had lessproliferation of sinusoidal cells, fewer mitotic figures and fewerchanges in the hepatocytes (e.g. vacuolation, presence of multiplenuclei, hepatocyte enlargement) than in the PBS treatment group. Thesedata demonstrate that mouse IL-28 and IL-29 have antiviral propertiesagainst a liver-trophic virus.

Example 40 LCMV Models

Lymphocytic choriomeningitis virus (LCMV) infections in mice mice are anexcellent model of acture and chronic infection. These models are usedto evaluate the effect of cytokines on the antiviral immune response andthe effects IL-28 and IL-29 have viral load and the antiviral immuneresponse. The two models used are: LCMV Armstrong (acute) infection andLCMV Clone 13 (chronic) infection. (See, e.g., Wherry et al., J. Virol.77:4911-4927, 2003; Blattman et al., Nature Med. 9(5):540-547, 2003;Hoffman et al., J. Immunol. 170:1339-1353, 2003.) There are three stagesof CD8 T cell development in response to virus: 1) expansion, 2)contraction, and 3) memory (acute model). IL-28 or IL-29 is injectedduring each stage for both acute and chronic models. In the chronicmodel, IL-28 or IL-29 is injected 60 days after infection to assess theeffect of IL-28 or IL-29 on persistent viral load. For both acute andchronic models, IL-28 or IL-29 is injected, and the viral load in blood,spleen and liver is examined. Other paramenter that can be examinedinclude: tetramer staining by flow to count the number of LCMV-specificCD8+ T cells; the ability of tetramer+cells to produce cytokines whenstimulated with their cognate LCMV antigen; and the ability ofLCMV-specific CD8+ T cells to proliferate in response to their cognateLCMV antigen. LCMV-specific T cells are phenotyped by flow cytometry toassess the cells activation and differentiation state. Also, the abilityof LCMV-specific CTL to lyse target cells bearing their cognate LCMVantigen is examined. The number and function of LCMV-specific CD4+ Tcells is also assessed.

A reduction in viral load after treatment with IL-28 or IL-29 isdetermined. A 50% reduction in viral load in any organ, especiallyliver, would be significant. For IL-28 or IL-29 treated mice, a 20%increase in the percentage of tetramer positive T cells thatproliferate, make cytokine, or display a mature phenotype relative tountreated mice would also be considered significant.

IL-28 or IL-29 injection leading to a reduction in viral load is due tomore effective control of viral infection especially in the chronicmodel where untreated the viral titers remain elevated for an extendedperiod of time. A two fold reduction in viral titer relative tountreated mice is considered significant.

Example 41 Influenza Model of Acute Viral Infection

A. Preliminary Experiment to Test Antiviral Activity

To determine the antiviral activity of IL-28 or IL-29 on acute infectionby Influenza virus, an in vivo study using influenza infected c57B1/6mice is performed using the following protocol:

Animals: 6 weeks-old female BALB/c mice (Charles River) with 148 mice,30 per group.

Groups:

Absolute control (not infected) to run in parallel for antibody titreand histopathology (2 animals per group)

Vehicle (i.p.) saline

Amantadine (positive control) 10 mg/day during 5 days (per os) starting2 hours before infection

IL-28 or IL-29 treated (5 μg, i.p. starting 2 hours after infection)

IL-28 or IL-29 (25 μg, i.p. starting 2 hours after infection)

IL-28 or IL-29 (125 μg, i.p. starting 2 hours after infection)

Day 0—Except for the absolute controls, all animals infected withInfluenza virus

For viral load (10 at LD50)

For immunology workout (LD30)

Day 0-9—daily injections of IL-28 or IL-29 (i.p.)

Body weight and general appearance recorded (3 times/week)

Day 3—sacrifice of 8 animals per group

Viral load in right lung (TCID50)

Histopathology in left lung

Blood sample for antibody titration

Day 10—sacrifice of all surviving animals collecting blood samples forantibody titration, isolating lung lymphocytes (4 pools of 3) for directCTL assay (in all 5 groups), and quantitative immunophenotyping for thefollowing markers: CD3/CD4, CD3/CD8, CD3/CD8/CD11b, CD8/CD44/CD62L,CD3/DX5, GR-1/F480, and CD19.

Study No. 2

Efficacy study of IL-28 or IL-29 in C57B1/6 mice infected withmouse-adapted virus is done using 8 weeks-old female C57B1/6 mice(Charles River).

Group 1: Vehicle (i.p.)

Group 2: Positive control: Anti-influenza neutralizing antibody (goatanti-influenza A/USSR (H1N1) (Chemicon International, Temecula, Calif.);40 μg/mouse at 2 h and 4 h post infection (10 μl intranasal)

Group 3: IL-28 or IL-29 (5 μg, i.p.)

Group 4: IL-28 or IL-29 (25 μg, i.p.)

Group 5: IL-28 or IL-29 (125 μg, i.p.)

Following-life observations and immunological workouts are prepared:

Day 0—all animals infected with Influenza virus (dose determined inexperiment 2)

Day 0-9—daily injections of IL-28 or IL-29 (i.p.)

Body weight and general appearance recorded every other day

Day 10—sacrifice of surviving animals and perform viral assay todetermine viral load in lung.

Isolation of lung lymphocytes (for direct CTL assay in the lungs usingEL-4 as targets and different E:T ratio (based on best results fromexperiments 1 and 2).

Tetramer staining: The number of CD8+ T cells binding MHC Class Itetramers containing influenza A nucleoprotein (NP) epitope are assessedusing complexes of MHC class I with viral peptides: FLU-NP₃₆₆₋₃₇₄/D^(b)(ASNENME™), (LMCV peptide/D^(b)).

Quantitative immunophenotyping of the following: CD8, tetramer,intracellular IFNγ, NK1.1, CD8, tetramer, CD62L, CD44, CD3(+or −),NK1.1(+), intracellular IFNγ, CD4, CD8, NK1.1 DX5, CD3 (+or −), NK1.1,DX5, tetramer, Single colour samples for cytometer adjustment.

Survival Re-Challenge Study

Day 30: Survival study with mice are treated for 9 days with differentdoses of IL-28 or IL-29 or with positive anti-influenza antibodycontrol. Body weight and antibody production in individual serum samples(Total, IgGI, IgG2a, IgG2b) are measured.

Re-Challenge Study:

Day 0: Both groups will be infected with A/PR virus (lLD30).

Group 6 will not be treated.

Group 7 will be treated for 9 days with 125 μg of IL-28 or IL-29.

Day 30: Survival study

Body weight and antibody production in individual serum samples (Total,IgGI, IgG2a, IgG2b) are measured.

Day 60: Re-challenge study

Survivors in each group will be divided into 2 subgroups

Group 6A and 7A will be re-challenge with A/PR virus (1 LD30)

Group 6B and 7B will be re-challenge with A/PR virus (1 LD30).

Both groups will be followed up and day of sacrifice will be determined.Body weight and antibody production in individual serum samples (Total,IgGI, IgG2a, IgG2b) are measured.

Example 42 IL-28 and IL-29 have Antiviral Activity Against Hepatitis BVirus (HBV) in vivo

A transgenic mouse model (Guidotti et al., J. Virology 69:6158-6169,1995) supports the replication of high levels of infectious HBV and hasbeen used as a chemotherapeutic model for HBV infection. Transgenic miceare treated with antiviral drugs and the levels of HBV DNA and RNA aremeasured in the transgenic mouse liver and serum following treatment.HBV protein levels can also be measured in the transgenic mouse serumfollowing treatment. This model has been used to evaluate theeffectiveness of lamivudine and IFN-α in reducing HBV viral titers.

HBV TG mice (male) are given intraperitoneal injections of 2.5, 25 or250 micrograms IL-28 or IL-29 every other day for 14 days (total of 8doses). Mice are bled for serum collection on day of treatment (day 0)and day 7. One hour following the final dose of IL-29 mice undergo aterminal bleed and are sacrificed. Serum and liver are analyzed forliver HBV DNA, liver HBV RNA, serum HBV DNA, liver HBc, serum Hbe andserum HBs.

Reduction in liver HBV DNA, liver HBV RNA, serum HBV DNA, liver HBc,serum Hbe or serum HBs in response to IL-28 or IL-29 reflects antiviralactivity of these compounds against HBV.

Example 43 IL-28 and IL-29 Inhibit Human Hemesvirus-8 (HHV-8)Replication in BCBL-1 Cells

The antiviral activities of IL-28 and IL-29 were tested against HHV-8 inan in vitro infection system using a B-lymphoid cell line, BCBL-1.

In the HHV-8 assay the test compound and a ganciclovir control wereassayed at five concentrations each, diluted in a half-log series. Theendpoints were TaqMan PCR for extracellular HHV-8 DNA (IC50) and cellnumbers using CellTiter96® reagent (TC50; Promega, Madison, Wis.).Briefly, BCBL-1 cells were plated in 96-well microtiter plates. After16-24 hours the cells were washed and the medium was replaced withcomplete medium containing various concentrations of the test compoundin triplicate. Ganciclovir was the positive control, while media alonewas a negative control (virus control, VC). Three days later the culturemedium was replaced with fresh medium containing the appropriatelydiluted test compound. Six days following the initial administration ofthe test compound, the cell culture supernatant was collected, treatedwith pronase and DNAse and then used in a real-time quantitative TaqManPCR assay. The PCR-amplified HHV-8 DNA was detected in real-time bymonitoring increases in fluorescence signals that result from theexonucleolytic degradation of a quenched fluorescent probe molecule thathybridizes to the amplified HHV-8 DNA. For each PCR amplification, astandard curve was simultaneously generated using dilutions of purifiedHHV-8 DNA. Antiviral activity was calculated from the reduction in HHV-8DNA levels (IC₅₀). A novel dye uptake assay was then employed to measurecell viability which was used to calculate toxicity (TC₅₀). Thetherapeutic index (TI) is calculated as TC₅₀/IC₅₀.

IL-28 and IL-29 inhibit HHV-8 viral replication in BCBL-1 cells. IL-28Ahad an IC₅₀ of 1 μg/ml and a TC₅₀ of >10 μg/ml (TI >10). IL-29 had anIC₅₀ of 6.5 μg/ml and a TC₅₀ of >10 μg/ml (TI>1.85). MetIL-29C172S-PEGhad an IC₅₀ of 0.14 μg/ml and a TC₅₀ of >10 μg/ml (TI>100).

Example 44 IL-28 and IL-29 Antiviral Activity Against Epstein Barr Virus(EBV)

The antiviral activities of IL-28 and IL-29 are tested against EBV in anin vitro infection system in a B-lymphoid cell line, P3HR-1. In the EBVassay the test compound and a control are assayed at five concentrationseach, diluted in a half-log series. The endpoints are TaqMan PCR forextracellular EBV DNA (IC50) and cell numbers using CellTiter96® reagent(TC50; Promega). Briefly, P3HR-1 cells are plated in 96-well microtiterplates. After 16-24 hours the cells are washed and the medium isreplaced with complete medium containing various concentrations of thetest compound in triplicate. In addition to a positive control, mediaalone is added to cells as a negative control (virus control, VC). Threedays later the culture medium is replaced with fresh medium containingthe appropriately diluted test compound. Six days following the initialadministration of the test compound, the cell culture supernatant iscollected, treated with pronase and DNAse and then used in a real-timequantitative TaqMan PCR assay. The PCR-amplified EBV DNA is detected inreal-time by monitoring increases in fluorescence signals that resultfrom the exonucleolytic degradation of a quenched fluorescent probemolecule that hybridizes to the amplified EBV DNA. For each PCRamplification, a standard curve was simultaneously generated usingdilutions of purified EBV DNA. Antiviral activity is calculated from thereduction in EBV DNA levels (IC₅₀). A novel dye uptake assay was thenemployed to measure cell viability which was used to calculate toxicity(TC₅₀). The therapeutic index (TI) is calculated as TC₅₀/IC₅₀.

Example 45 IL-28 and IL-29 Antiviral Activity Against Herpes SimplexVirus-2 (HSV-2)

The antiviral activities of IL-28 and IL-29 were tested against HSV-2 inan in vitro infection system in Vero cells. The antiviral effects ofIL-28 and IL-29 were assessed in inhibition of cytopathic effect assays(CPE). The assay involves the killing of Vero cells by the cytopathicHSV-2 virus and the inhibition of cell killing by IL-28 and IL-29. TheVero cells are propagated in Dulbecco's modified essential medium (DMEM)containing phenol red with 10% horse serum, 1% glutamine and 1%penicillin-streptomycin, while the CPE inhibition assays are performedin DMEM without phenol red with 2% FBS, 1% glutamine and 1% Pen-Strep.On the day preceding the assays, cells were trypsinized (1%trypsin-EDTA), washed, counted and plated out at 10⁴ cells/well in a96-well flat-bottom BioCoat® plates (Fisher Scientific, Pittsburgh, Pa.)in a volume of 100 μl/well. The next morning, the medium was removed anda pre-titered aliquot of virus was added to the cells. The amount ofvirus used is the maximum dilution that would yield complete cellkilling (>80%) at the time of maximal CPE development. Cell viability isdetermined using a CellTiter 96® reagent (Promega) according to themanufacturer's protocol, using a Vmax plate reader (Molecular Devices,Sunnyvale, Calif.). Compounds are tested at six concentrations each,diluted in assay medium in a half-log series. Acyclovir was used as apositive control. Compounds are added at the time of viral infection.The average background and drug color-corrected data for percent CPEreduction and percent cell viability at each concentration aredetermined relative to controls and the IC₅₀ calculated relative to theTC₅₀.

IL-28A, IL-29 and MetIL-29C172S-PEG did not inhibit cell death (IC₅₀of >10 ug/ml) in this assay. There was also no antiviral activity ofIFN␣ in the assay.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (e.g., GenBank aminoacid and nucleotide sequence submissions) cited herein are incorporatedby reference. The foregoing detailed description and examples have beengiven for clarity of understanding only. No unnecessary limitations areto be understood therefrom. The invention is not limited to the exactdetails shown and described, for variations obvious to one skilled inthe art will be included within the invention defined by the claims.

1. An antibody or antibody fragment that specifically binds to apolypeptide comprising amino acid residues 1-175 of SEQ ID NO:127. 2.The antibody of claim 1, wherein the antibody is selected from the groupconsisting of a polyclonal antibody, a murine monoclonal antibody, ahumanized antibody derived from a murine monoclonal antibody, anantibody fragment, neutralizing antibody, and a human monoclonalantibody.
 3. The antibody fragment of claim 1, wherein the antibodyfragment is selected from the group consisting of F(ab′), F(ab), Fab′,Fab, Fv, scFv, and minimal recognition unit.